G.P. is a Marie Sk&#322;odowska-Curie Fellow funded by the European Union MSCA-IF-2014 project Re.B.Us, G.A. n.660689, under the framework program H2020. The GUI mapMEA is freely available upon request to author ilaria.colombi@iit.it.

<ol>
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(92), (2014).</li><li>Boido, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortico-hippocampal+hyperexcitability+in+synapsin+I/II/III+knockout+mice:+age-dependency+and+response+to+the+antiepileptic+drug+levetiracetam.">Cortico-hippocampal hyperexcitability in synapsin I/II/III knockout mice: age-dependency and response to the antiepileptic drug levetiracetam.</a> Neuroscience. 171 (1), 268-283 (2010).</li><li>Gonzalez-Sulser, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+4-aminopyridine+in+vitro+epilepsy+model+analyzed+with+a+perforated+multi-electrode+array.">The 4-aminopyridine in vitro epilepsy model analyzed with a perforated multi-electrode array.</a> Neuropharmacology. 60 (7-8), 1142-1153 (2011).</li><li>Hajos, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintaining+network+activity+in+submerged+hippocampal+slices:+importance+of+oxygen+supply.">Maintaining network activity in submerged hippocampal slices: importance of oxygen supply.</a> Eur J Neurosci. 29 (2), 319-327 (2009).</li><li>Ivanov, A., Zilberter, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+state+of+energy+metabolism+in+brain+slices:+the+principal+role+of+oxygen+delivery+and+energy+substrates+in+shaping+neuronal+activity.">Critical state of energy metabolism in brain slices: the principal role of oxygen delivery and energy substrates in shaping neuronal activity.</a> Front Neuroenergetics. 3, 9 (2011).</li><li>MultichannelSystems. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual PH01. , (2018).</li><li>Zhang, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Propagation+of+epileptiform+activity+can+be+independent+of+synaptic+transmission,+gap+junctions,+or+diffusion+and+is+consistent+with+electrical+field+transmission.">Propagation of epileptiform activity can be independent of synaptic transmission, gap junctions, or diffusion and is consistent with electrical field transmission.</a> J Neurosci. 34 (4), 1409-1419 (2014).</li><li>Panuccio, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+ictogenesis+and+parahippocampal+networks+in+a+rodent+model+of+temporal+lobe+epilepsy.">In vitro ictogenesis and parahippocampal networks in a rodent model of temporal lobe epilepsy.</a> Neurobiol Dis. 39 (3), 372-380 (2010).</li><li>Barbarosie, M., Avoli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CA3-driven+hippocampal-entorhinal+loop+controls+rather+than+sustains+in+vitro+limbic+seizures.">CA3-driven hippocampal-entorhinal loop controls rather than sustains in vitro limbic seizures.</a> J Neurosci. 17 (23), 9308-9314 (1997).</li><li>Panuccio, G., Guez, A., Vincent, R., Avoli, M., Pineau, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adaptive+control+of+epileptiform+excitability+in+an+in+vitro+model+of+limbic+seizures.">Adaptive control of epileptiform excitability in an in vitro model of limbic seizures.</a> Exp Neurol. 241, 179-183 (2013).</li><li>Benini, R., Avoli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rat+subicular+networks+gate+hippocampal+output+activity+in+an+in+vitro+model+of+limbic+seizures.">Rat subicular networks gate hippocampal output activity in an in vitro model of limbic seizures.</a> J Physiol. 566 (Pt 3), 885-900 (2005).</li><li>D'Arcangelo, G., Panuccio, G., Tancredi, V., Avoli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repetitive+low-frequency+stimulation+reduces+epileptiform+synchronization+in+limbic+neuronal+networks.">Repetitive low-frequency stimulation reduces epileptiform synchronization in limbic neuronal networks.</a> Neurobiol Dis. 19 (1-2), 119-128 (2005).</li><li>Steidl, E. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spreading+depolarization+in+the+brainstem+mediates+sudden+cardiorespiratory+arrest+in+mouse+SUDEP+models.">Spreading depolarization in the brainstem mediates sudden cardiorespiratory arrest in mouse SUDEP models.</a> Sci Transl Med. 7 (282), 282ra246 (2015).</li><li>MA, R., Avoli, M., Noebels, J. L., Rogawski, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Jasper's Basic Mechanisms of the Epilepsies . , (2012).</li><li>Motamedi, G. 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The authors have nothing to disclose.

MEAs are an invaluable tool for neurophysiology investigations and have reached maturity in recent years thanks to decades of exploration and development. As compared to conventional field potential recording, MEAs offer the great advantage of a higher number of observation points and known inter-electrode distance, which are crucial to accurately pinpoint neuronal network interactions.
The MEA recording technique can also be coupled to other electrophysiology approaches, such as patch-clamp recording15, to investigate the relation between single neuron and neuronal network activity. Moreover, the possibility of visualizing field potentials and multi-unit activity simultaneously can provide precious insights into the correlation between the activity of small neuronal ensembles and collective neuronal networks. The combination with voltage-sensitive dyes, calcium imaging, and optogenetics29 allows inferring neurophysiology phenomena using polyhedral approaches. In addition to pharmacological studies, the possibility of performing both recording and stimulation with the same system makes the MEA recording technique very powerful and versatile: for example, it is possible to study the synaptic plasticity phenomena at the core of memory formation30, using the neuromodulation protocols presented here, which are relevant to DBS to treat epilepsy and a wide variety of brain disorders. The recent proof that the MEA recording technique can be successfully applied to human epileptic brain tissue16 demonstrates its invaluable usefulness in epilepsy research, both to understand the basic mechanisms underlying this devastating disease and to fine-tune DBS algorithms to ameliorate it.
However, MEAs force the pursuit of electrophysiology experiments under &#39;limit&#39; conditions for brain slices, due to the requirement of a submerged recording chamber and the necessity of letting the brain slice rest on the solid substrate where the micro-electrodes are integrated. Thus, brain tissue may not receive adequate oxygen supply, which in turn may affect the quality of the recordings.
The protocol described here allows to reliably record and study ictogenesis in rodent limbic networks coupled to MEAs using the acute in vitro 4AP model and to pursue prolonged epochs of electrical stimulation, which provide relevant information for the evaluation of DBS policies.
Previous studies on electrical neuromodulation of epileptic limbic networks based on the use of conventional field potential recording have used either mouse or rat brain slices with similar results24,25; but the use of rat brain tissue proved more challenging, reasonably due to the weaker connectivity as compared to brain tissue obtained from mice7. With regard to the MEA technique, mouse brain slices are best suited by virtue of their smaller size. Moreover, given the higher rate of occurrence of ictal-like discharges in mouse versus rat brain tissue, it is possible to test significantly more brain slices throughout one experimental day, which in turn makes data collection faster and more efficient, and can reduce the number of animals used.
Significance with Respect to Existing Methods:
Ictal discharges recorded using the custom chamber described here appear to be similar in duration and rate of occurrence to those observed using the conventional MEA ring chamber and the same MEA type (planar micro-electrodes, cf. 17). However, it needs to be emphasized that brain slice thickness must be significantly reduced when using the conventional round recording chamber along with a low perfusion rate (1 mL/min), which may hinder the successful pursuit of neuromodulation experiments due to poor connectivity.
In this protocol, a low-volume custom recording chamber inspired by patch-clamp recording chamber design provides stable and reliable laminar flow that is crucial for the successful pursuit of MEA recordings; it also allows increasing the brain slice thickness to 400 &#181;m in order to achieve a fair trade-off between tissue viability and intrinsic connectivity. It is indeed recognized that laminar flow of the recording solution within the chamber is highly desirable for brain slice electrophysiology, since it is not affected by temperature, oxygen, and pH gradients that are observed in circular-flow round recording chambers19,20,31 (like the ones provided with commercially available MEA). Such gradients introduce experimental bias and are also detrimental to the brain slice. Recording chambers of a relatively small volume (~1.5 mL) along with a high perfusion rate (5 -&#160;6 mL/min)16,31 allow for adequate exchange of the perfusion medium (&#8805;3 times/min). The custom chamber can be easily obtained from commercial sources at an affordable price or produced in-house using 3D printing technology. Other studies have reported MEA recordings from 400 &#181;m-thick human brain slices16 using the conventional MEA ring chamber, while keeping the ACSF volume to 1 mL and enforcing a high perfusion rate (5 - 6 mL/min) with the help of a low-noise peristaltic pump. However, the authors have used a different model of ictogenesis, i.e., the low Mg2+, which is likely less influenced than the 4AP model by ephaptic mechanisms involving significant increases in extracellular K+ concentration11,22. We have found that a high perfusion rate is not desirable in the 4AP model, possibly due to the fast wash out of accumulated extracellular K+, which may need to be significantly increased in the recording ACSF16. In fact, ictal events appeared more &#8216;chunked&#8217; when ASCF perfusion speed was increased to 2 - 3 mL/min and the brain slice was submerged by a thick ACSF layer, whereas full-blown discharges exhibiting robust tonic-clonic components could be restored upon the return to a lower perfusion rate (1 mL/min) and a thinner ACSF layer right at the tissue surface level (data not shown). The custom recording chamber described in this protocol allows exchanging the perfusion medium 3 - 5 times/min at a flow rate of 1 mL/min. Thus, the overall oxygen supply to the brain slices is strongly improved even at relatively low perfusion rates, while still guaranteeing a stable recording temperature and high signal-to-noise ratio. Most importantly, it is possible to keep the extracellular K+ concentration to a physiological value.
The 4AP model of ictogenesis does not require any significant modification of the ACSF ionic composition, such as lowering Mg2+ or increasing K+, and offers the unique advantage of keeping both excitatory and inhibitory transmissions intact12, an aspect that is highly relevant in epilepsy research in light of the crucial role of both glutamatergic and GABAergic networks in epileptiform synchronization (cf. 7). The 4AP-ACSF used in this protocol contains a physiological K+ concentration (3.25 mM) and a slightly lower Mg2+ concentration than the holding ACSF (1 mM versus 1.3 mM). This concentration still falls within the reported physiological values of Mg2+ concentration in the rodent CSF and it is used in many laboratories (see for example17,18). To the purpose of the described protocol, we have found that this slight decrease is preferred to aid in the effect of 4AP.
In previous work, 4AP has been applied to the brain slice when it was already positioned inside the recording chamber17,18. Unless the experimenter needs to observe the time latency between the 4AP application and the onset of epileptiform patterns, we find that this approach is rather time-consuming and is not suitable to pursue the prolonged recording and stimulation sessions described in this protocol. Pre-incubation of brain slices in 4AP at 32 &#176;C allows sparing much experimental time, since brain slices can be pre-treated in series while pursuing the experiment using other tissue sections.
The prolonged viability of brain slices may be useful to analyze network features in the long-term. Moreover, their improved resilience to repetitive electrical stimulation is of great advantage if the experimenter wants to compare several stimulation protocols, which must be performed in the same brain slice for statistical robustness. In our hands, when testing 3 stimulation protocols, each preceded by a control phase and followed by a recovery phase, the experiment may last 3 - 5 h. In this context, live MEA mapping is crucial in order to activate the correct pathway and suppress rather than favor ictogenesis via electrical stimulation. Our user-friendly GUI represents a quick, simple and flexible tool to map the electrodes of different brain structures. Contrary to commercial software, it is possible to add custom MEA layouts even with basic programming skills. Images can be acquired in bright field; thus, any general purpose camera that has good resolution and fits on a microscope is suitable. An inverted microscope is essential to visualize the microelectrodes position relative to the brain slice, since these would be hidden underneath the tissue if using an upright microscope. However, a stereo-microscope is recommended in addition to the inverted type if the experimenter needs to perform knife-cuts to disrupt specific neuronal pathways.
Finally, a further advantage of the proposed approach is the cheap and relatively easy assembly of most of the required tools, like the custom recording chamber and the holding chambers, the warm bath and the slice anchor, as well as the unnecessary use of a costly low-noise peristaltic pump.
Limitations of the Technique:
MEA recording does not allow visualizing very slow waves, i.e., DC shifts in the signal. Such baseline deflections may help asymptotic measurement of ictal discharge duration and, most important, they are fundamental to study cortical spreading depression (a phenomenon that relates to sudden unexpected death in epilepsy32 and is shared between epilepsy and migraine33).
With regard to electrical neuromodulation, the protocol described here allows performing several stimulation sessions without affecting brain slice viability. We could successfully perform up to 3 stimulation sessions of 20 - 45 min, similar to what reported in previous work25. Although brain slices may probably withstand a higher number of stimulation sessions or longer stimulation protocols, we did not test brain slices in this regard. We recommend limiting the number of stimulation protocols to 3 and avoiding prolonged (&#8805;60 min) stimulation sessions, which may significantly stress brain tissue maintained under these limit conditions, up to the lack of recovery upon stimulus withdrawal.
Critical Steps Within the Protocol:
Several critical factors may hinder the successful pursuit of recording and modulation of the epileptiform activity in brain slices coupled to MEA. Besides the rodent species used and the quality of the brain slices, the perfusion rate during recording and the dynamics of the ACSF flow (i.e., laminar versus circular) within the recording chamber, we have found that the conditions of recovery and long-term maintenance of brain slices as well as the mode of 4AP application are the most critical steps.
When using a submerged holding chamber it is crucial to store the brain slices at room temperature to preserve brain tissue network activity and favor the induction of ictal-like discharges by 4AP application. In our hands, recovery and maintenance at 32 &#176;C deteriorated the brain tissue within 3 - 4 h of slicing.
Incubation of brain slices in 4AP-ACSF at room temperature and recording at 32 &#176;C is, however, not desirable. We have found many difficulties in observing robust recurrent ictal discharges in this condition. Indeed, low temperatures in the 20&#8211;24 &#176;C range have been reported to dampen or even prevent 4AP-induced ictogenesis both in vitro34 and in vivo35. Thus, the 4AP incubation and recording temperatures must fall within the 30 - 34 &#176;C and must be matched. To avoid putting the brain tissue under too much stress due to the simultaneous induction of hyperexcitability by 4AP and the sudden exposure of brain slices from room temperature to the warm (32 &#176;C) 4AP-ACSF, it is fundamental to perform an intermediate pre-warming step and let the brain slices habituate in the holding ACSF at 32 &#176;C for 20 - 30 min. Skipping this step may affect ictogenesis induction by 4AP.
Another aspect that needs to be mentioned is the importance of D-glucose concentration in the ACSF after slicing. A 25 mM concentration preserves the brain slices better than the 10 mM concentration used in many laboratories and it also improves the rate of occurrence of ictal activity, which is 2-fold faster (thus, making it easier to pursue a long series of experimental protocols in several brain slices each day).
Lastly, some important fine details during the slicing procedure need to be mentioned. First, do not allow the the intact brain and the brain slices to freeze in the cold cutting ACSF. Chilling the brain for &#60;2 min is sufficient given the small size of the mouse brain. Tissue sections should be transferred immediately from the buffer tray to the rinsing beakers at room temperature. Rinsing the cutting ACSF is very important otherwise the high sucrose concentration will make the brain slices stick to the nylon mesh of the holding chamber. To effectively rinse the tissue, it is important to minimize the cutting ACSF content within the transfer pipette so as to not contaminate the holding ACSF excessively. The 2-stage rinsing aids in minimizing contamination. Upon completion of the slicing procedure, tissue sections should be transferred immediately from the rinsing beakers to the holding chamber, where the soft nylon mesh suspended half-way in the holding ACSF allows tissue oxygenation on both sides. The same principle should be applied at all stages following the slicing procedure: brain slices should never sit at the bottom of the beaker, they should not be in contact with the sides of the holding chambers, and they should never be in contact with each other nor overlap.
Modifications and Troubleshooting:
ACSF composition may be modified according to the experimenter&#39;s needs. For example, drugs may be added to dissect the contribution of specific neurotransmitters or ion channels to the efficacy of electrical neuromodulation. Moreover, pyruvic and ascorbic acid (cf. Table 2) may be omitted, although we find that they exert a powerful neuroprotective role. We report in Table 3 the most common issues that can be encountered in this protocol and how to deal with them.
Conclusions:
MEA recording is undoubtedly an invaluable technique to address the interactions of neuronal networks in health and disease. In addition to pharmacological studies, it is also possible to evaluate electrical neuromodulation protocols that are relevant to DBS applied to epilepsy and other neurological disorders. In this protocol, we have shown that it is possible to reproduce typical periodic stimulation experiments with similar results to those obtained with conventional extracellular field potential recording and external stimulating electrodes. The increasing availability of commercial user-friendly equipment and advanced software tools make the MEA recording technique also suitable for closed-loop stimulation experiments, to provide ad hoc stimulation to the brain tissue, and to investigate the contribution of feedback mechanisms to neuronal network responses.

Epilepsy is a life-threatening progressive disorder causing uncontrolled activity of the brain1; it carries among the highest burden of disease and significant social stigma2,3. TLE is the most frequent syndrome (40%) and the most frequently (~ 30%) resistant to anti-epileptic medications4. While surgical ablation of the epileptogenic tissue may ameliorate the patient&#39;s condition, it is not feasible in all patients and may not guarantee a completely seizure-free life5. Modulation of epileptic limbic networks by electrical DBS is a promising approach when pharmacological treatment or neurosurgery are not suitable.
Rodent brain slices are a valuable tool to study how neuronal networks function in health and disease6 by means of in vitro electrophysiology techniques, as they preserve, at least in part, the original architecture and connectivity of the brain region(s) of interest (ROI). In particular, horizontal combined hippocampus-entorhinal cortex (hippocampus-EC) slices comprise the essential neuronal networks involved in TLE and are therefore routinely employed in in vitro TLE research7.
Seizure-like activity may be acutely induced in brain slices by changing the ionic composition of the artificial cerebrospinal fluid (ACSF), such as decreasing magnesium while increasing potassium8,9,10, or by means of pharmacological manipulations, such as blocking inhibitory GABAergic activity (see11 for a comprehensive review). However, these models are based on the unbalanced alteration of excitation and inhibition; thus, they do not allow studying the interaction and concerted contribution of excitatory and inhibitory networks to ictogenesis. Continuous perfusion of brain slices with the convulsant drug 4AP enhances both excitatory and inhibitory neurotransmission, thus allowing to study acute ictogenesis while keeping synaptic activity overall intact12.
MEAs allow recording the electrical activity generated by neuronal networks from a greater number of observation points as compared to conventional extracellular field potential recording, where spatial restrictions limit the number of electrodes that can be accommodated on the brain slice surface. Moreover, MEA chips offer the added advantage of known inter-electrode distance, which is very useful to trace propagation and assess the traveling velocity of recorded signals. Although initially conceived for recording from cultured neurons13,14, MEAs are now also used to characterize the electrophysiological features of acute brain slices obtained from rodents15 and humans16. Thus, in the context of epilepsy research, MEAs represent a valuable tool to pinpoint neuronal networks&#39; interactions at the core of ictogenesis16,17,18.
However, MEA recording inherently carries technical challenges in obtaining or maintaining a stable epileptiform pattern throughout long (several hours) experimental protocols. First, tissue oxygenation may not be adequate within the large and round submerged-type recording chamber19,20 typical of commercially available MEAs, while poor signal-to-noise ratio and temperature instability might affect the quality of the recording when a high perfusion rate (6 - 10 mL/min) is used to improve the oxygen supply to the brain slice (see for example the technical notes on heating and perfusion equipment21). Second, recurrent discharges featuring high frequency components, such as epileptiform discharges, are hardly observed when using submerged-type recording chambers19; this is particularly the case when the acute epileptiform pattern is chemically induced and involves ephaptic mechanisms, as it is the case for the 4AP model22 (see11 for a comprehensive overview). To overcome these limitations, several strategies have been proposed by researchers. For example, increasing the perfusion rate19,20 while reducing brain slice thickness (&#8804;300 &#181;M) and area17,18 are crucial factors to achieving adequate oxygen supply to the tissue. In addition, improved brain slice viability can also be accomplished by using perforated MEAs (pMEAs), which allow perfusing the brain tissue from both sides18.
Whereas the described approaches have significantly improved the feasibility of MEA recording from brain slices, they have not been tested against prolonged (several hours) recording and electrical stimulation sessions, the latter representing a significant stress for the brain tissue. Prolonged recording sessions may be required to study the evolution in time of specific features of epileptiform patterns that would not be possible to unmask by short-term measurements. In the context of DBS research, prolonged experimental protocols may be required to evaluate and compare the effects of several stimulation paradigms in the same brain slice.
When only spontaneous electrical activity needs to be recorded, mapping of the electrodes with respect to the ROI is usually done a posteriori, i.e., during data analysis; instead, the study of evoked responses or of electrical neuromodulation paradigms requires that stimulation be delivered to specific ROI(s), dictating the need of quick and easy live electrode mapping during the experiment.
Here, we illustrate a simple experimental protocol that allows for induction and maintenance of a stable 4AP-induced epileptiform pattern in adult rodent brain slices. The observed activity faithfully reproduces the electrophysiological features of this model as characterized using conventional extracellular electrophysiology techniques. It persists for several hours and outlasts sustained electrical stimulation for repeated prolonged epochs. The chambers used to maintain and incubate the brain slices can be easily assembled using standard laboratory supplies (Figure 1A), whereas a custom recording chamber allowing for optimal solution exchange rate and laminar flow (Figure 1B) can be obtained from commercial sources or using 3D printing technology at an affordable price. Quick mapping of the ROI(s) for real-time monitoring and for the selection of stimulating electrodes is made possible by a custom user-friendly GUI named mapMEA, freely available upon request.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57548/57548fig1.jpg" /> 
Figure 1: Custom equipment used for this protocol. (A) The holding chambers for recovery, pre-warming, and pre-incubation in 4AP are assembled using a beaker and a Petri dish. The Petri dish should be smaller in diameter than the breaker and is held in place by means of a syringe plunger. The bottom of the Petri dish is replaced by a nylon mesh (from soft stockings) glued with cyanoacrylate onto the rim of the Petri dish. Oxygen is supplied via a bent spinal needle (22 G), inserted between the walls of the Petri dish and the beaker. Bubbles should be emerging from the side of the beaker and never reach the nylon mesh to avoid brain slice displacement. The top of a Petri dish can be used as a lid to avoid ACSF evaporation and maintain oxygen saturation. (B) Custom recording chamber. in: inlet reservoir to accommodate the heating cannula and the reference electrode. out: outlet reservoir to accommodate the suction needle. rec:recording chamber. (C) Glass Pasteur pipette, curled by fire-polishing, used to handle the tissue and adjust its position within the recording chamber. (D) Custom slice hold-down anchor. (E) Final assembly of the recording chamber mounted onto the MEA chip. A brain slice rests onto the bottom held in place by the anchor. The red arrow indicates the PTFE tubing covering the heating cannula, whereas the red circle indicates the reference electrode, a saturated KCl pellet submerged by ACSF in the inlet reservoir. The blue arrow indicates the suction needle in the outlet reservoir. <a href="/files/ftp_upload/57548/57548fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>Poly-D-Lysine&amp;nbsp;</td><td>Sigma-Adrich</td><td>P7886</td><td>Needed for MEA coating</td></tr><tr><td>NaCl</td><td>Sigma-Adrich</td><td>S9888</td><td>Chemical</td></tr><tr><td>KCl</td><td>Sigma-Adrich</td><td>P9541</td><td>Chemical</td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma-Adrich</td><td>795488</td><td>Chemical</td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;*2 H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Adrich</td><td>C3306</td><td>Chemical</td></tr><tr><td>D-Glucose</td><td>Sigma-Adrich</td><td>RDD016</td><td>Chemical</td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Adrich</td><td>S5761</td><td>Chemical</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;*6 H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Adrich</td><td>M2670</td><td>Chemical</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;*6 H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma-Adrich</td><td>M5921</td><td>Chemical</td></tr><tr><td>Sucrose</td><td>Sigma-Adrich</td><td>RDD023</td><td>Chemical</td></tr><tr><td>Pyruvic Acid, 98%&amp;nbsp;</td><td>Sigma-Adrich</td><td>107360</td><td>Chemical</td></tr><tr><td>4-aminopyridine</td><td>Sigma-Adrich</td><td>A78403</td><td>Convulsant drug</td></tr><tr><td>STG-2004</td><td>Multichannel System</td><td>STG4004-1.6mA</td><td>4-channel stimulus generator with voltage (&amp;plusmn;8 V) and current output (&amp;plusmn;1.6 mA)</td></tr><tr><td>MEA1060</td><td>Multichannel System</td><td>N/A</td><td>MEA amplifier</td></tr><tr><td>planar MEA</td><td>Multichannel System</td><td>60MEA500/30iR-Ti w/o ring</td><td>Must be without ring to allow using the custom recordingchamber</td></tr><tr><td>McRack</td><td>Multichannel System</td><td></td><td>Recording software</td></tr><tr><td>McStimulus II</td><td>Multichannel System</td><td></td><td>STG4004 control software</td></tr><tr><td>TC02</td><td>Multichannel System</td><td>TC02</td><td>2-channel thermostat</td></tr><tr><td>PH01</td><td>Multichannel System</td><td>PH01</td><td>Heating perfusion canula</td></tr><tr><td>MPH&amp;nbsp;</td><td>Multichannel System</td><td>MPH&amp;nbsp;</td><td>Magnetic holder for PH01 and suction needle</td></tr><tr><td>Elastostil E43</td><td>Wacker</td><td>E43</td><td>Elastomeric sealant used to mount the custom recording chamber onto the MEA</td></tr><tr><td>MEA Custom Chamber</td><td>Crisel Instrument</td><td>SKE-chamber MEA</td><td>Custom recording chamber</td></tr><tr><td>Ag/AgCl electrode, pellet, 1.0 mm&amp;nbsp;</td><td>Crisel Instrument</td><td>64-1309</td><td>Reference electrode for the custom recording chamber</td></tr><tr><td>Tergazyme</td><td>Sigma-Adrich</td><td>Z273287-1EA</td><td>Enzymatic cleaner</td></tr><tr><td>MATLAB</td><td>The Mathworks</td><td></td><td>Programming environment for electrodemapping</td></tr><tr><td>VT1000S</td><td>Leica Biosystems</td><td>VT1000S</td><td>Vibratome</td></tr><tr><td></td><td>Warner Instruments</td><td>64-1309</td><td>Ag-AgCl Electrode Pellet 1.0 mm (E205). Reference electrode.</td></tr></tbody></table>

recording and modulation of epileptiform activity in rodent brain slices coupled to microelectrode arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

Watch this Scientific Journal Video about Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays at JoVE.com

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

recording-modulation-epileptiform-activity-rodent-brain-slices

Research

JoVE Journal

Neuroscience

14.6K Views.  Istituto Italiano di Tecnologia. We illustreren hoe u opnemen en elektrische modulatie van 4-aminopyridine-geïnduceerde epileptiform activiteit in knaagdier hersenen segmenten met behulp van micro-elektrode arrays. Een aangepaste opname-kamer onderhoudt weefsel levensvatbaarheid in langdurige experimentele sessies. Leven van de elektrode toewijzing en selectie van stimulerende paren worden uitgevoerd door een aangepaste grafische gebruikersinterface.

Video: Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Multi-electrode Array Recordings of Human Epileptic Postoperative Cortical Tissue

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which disrupt normal brain function. Despite treatment with currently available antiepileptic drugs targeting neuronal functions, one third of patients with epilepsy are pharmacoresistant. In this condition, surgical resection of the brain area generating seizures remains the only alternative treatment. Studying human epileptic tissues has contributed to understand new epileptogenic mechanisms during the last 10 years. Indeed, these tissues generate spontaneous interictal epileptic discharges as well as pharmacologically-induced ictal events which can be recorded with classical electrophysiology techniques. Remarkably, multi-electrode arrays (MEAs), which are microfabricated devices embedding an array of spatially arranged microelectrodes, provide the unique opportunity to simultaneously stimulate and record field potentials, as well as action potentials of multiple neurons from different areas of the tissue. Thus MEAs recordings offer an excellent approach to study the spatio-temporal patterns of spontaneous interictal and evoked seizure-like events and the mechanisms underlying seizure onset and propagation. Here we describe how to prepare human cortical slices from surgically resected tissue and to record with MEAs interictal and ictal-like events ex vivo.

We here describe how to perform multi-electrode array recordings of human epileptic cortical tissue. Epileptic tissue resection, slice preparation and multi-electrode array recordings of interictal and ictal events are demonstrated in detail.

Epilepsy, affecting about 1% of the population, comprises a group of neurological disorders characterized by the periodic occurrence of seizures, which ...

Medicine

Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the stimulation parameters (e.g., orientation, field strength, and stimulation duration) need to be examined in mice brain slice preparations. Testing and arranging the orientation of the electrode relative to the position of the mice brain slice are feasible. The present method preserves the thalamocingulate pathway to evaluate the effect of DCS on anterior cingulate cortex seizure-like activities. The results of the multichannel array recordings indicated that cathodal DCS significantly decreased the amplitude of the stimulation-evoked responses and duration of 4-aminopyridine and bicuculline-induced seizure-like activity. This study also found that cathodal DCS applications at 15 min caused long-term depression in the thalamocingulate pathway. The present study investigates the effects of DCS on thalamocingulate synaptic plasticity and acute seizure-like activities. The current procedure can test the optimal stimulation parameters including orientation, field strength, and stimulation duration in an in vitro mouse model. Also, the method can evaluate the effects of DCS on cortical seizure-like activities at both the cellular and network levels.

Studies have shown that cathodal transcranial direct-current stimulation can produce suppressive effects on drug-resistant seizures. In this study, an in vitro experimental setup was devised in which the direct-current stimulation and multielectrode array recording of seizure-like activity were evaluated in mice brain slice preparation. The direct-current stimulation parameters were evaluated.

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the ...

Recording Network Activity in Spinal Nociceptive Circuits Using Microelectrode Arrays

The roles and connectivity of specific types of neurons within the spinal cord dorsal horn (DH) are being delineated at a rapid rate to provide an increasingly detailed view of the circuits underpinning spinal pain processing. However, the effects of these connections for broader network activity in the DH remain less well understood because most studies focus on the activity of single neurons and small microcircuits. Alternatively, the use of microelectrode arrays (MEAs), which can monitor electrical activity across many cells, provides high spatial and temporal resolution of neural activity. Here, the use of MEAs with mouse spinal cord slices to study DH activity induced by chemically stimulating DH circuits with 4-aminopyridine (4-AP) is described. The resulting rhythmic activity is restricted to the superficial DH, stable over time, blocked by tetrodotoxin, and can be investigated in different slice orientations. Together, this preparation provides a platform to investigate DH circuit activity in tissue from na&#239;ve animals, animal models of chronic pain, and mice with genetically altered nociceptive function. Furthermore, MEA recordings in 4-AP-stimulated spinal cord slices can be used as a rapid screening tool to assess the capacity of novel antinociceptive compounds to disrupt activity in the spinal cord DH.

The combined use of microelectrode array technology and 4-aminopyridine-induced chemical stimulation for investigating network-level nociceptive activity in the spinal cord dorsal horn is outlined.

The roles and connectivity of specific types of neurons within the spinal cord dorsal horn (DH) are being delineated at a rapid rate to provide an ...

Recording Network Activity in Brain Assembloids: Multi-Electrode Array Approach

A Multi-Electrode Array Platform for Modeling Epilepsy Using Human Pluripotent Stem Cell-Derived Brain Assembloids

Human brain organoids are three-dimensional (3D) structures derived from human pluripotent stem cells (hPSCs) that recapitulate&#160;aspects of fetal brain development. The fusion of dorsal with ventral regionally specified brain organoids in vitro generates assembloids, which have functionally integrated microcircuits with excitatory and inhibitory neurons. Due to their structural complexity and diverse population of neurons, assembloids have become a useful in vitro tool for studying aberrant network activity. Multi-electrode array (MEA) recordings serve as a method for capturing electrical field potentials, spikes, and longitudinal network dynamics from a population of neurons without compromising cell membrane integrity. However, adhering assembloids onto the electrodes for long-term recordings can be challenging due to their large size and limited contact surface area with the electrodes. Here, we demonstrate a method to plate assembloids onto MEA plates for recording electrophysiological activity over a 2-month span. Although the current protocol utilizes human cortical organoids, it can be broadly adapted to organoids differentiated to model other brain regions. This protocol establishes a robust, longitudinal, electrophysiological assay for studying the development of a neuronal network, and this platform has the potential to be used in drug screening for therapeutic development in epilepsy.

Author Spotlight: Advancing Genetic Epilepsy Studies with Multi-Electrode Array-Based Long-Term Electrophysiological Monitoring of Human Brain Assembloids

This protocol aims to stably plate dorsal-ventral fused assembloids on multi-electrode arrays for modeling epilepsy in vitro.

Human brain organoids are three-dimensional (3D) structures derived from human pluripotent stem cells (hPSCs) that recapitulate&#160;aspects of fetal ...

Consistent Induction and Recording of Seizure-Like Activity Using CMOS-HD-MEAs

High-Quality Seizure-Like Activity from Acute Brain Slices Using a Complementary Metal-Oxide-Semiconductor High-Density Microelectrode Array System

Complementary metal-oxide-semiconductor high-density microelectrode array (CMOS-HD-MEA) systems can record neurophysiological activity from cell cultures and ex vivo brain slices in unprecedented electrophysiological detail. CMOS-HD-MEAs were first optimized to record high-quality neuronal unit activity from cell cultures but have also been shown to produce quality data from acute retinal and cerebellar slices. Researchers have recently used CMOS-HD-MEAs to record local field potentials (LFPs) from acute, cortical rodent brain slices. One LFP of interest is seizure-like activity. While many users have produced brief, spontaneous epileptiform discharges using CMOS-HD-MEAs, few users reliably produce quality seizure-like activity. Many factors may contribute to this difficulty, including electrical noise, the inconsistent nature of producing seizure-like activity when using submerged recording chambers, and the limitation that 2D CMOS-MEA chips only record from the surface of the brain slice. The techniques detailed in this protocol should enable users to consistently induce and record high-quality seizure-like activity from acute brain slices with a CMOS-HD-MEA system. In addition, this protocol outlines the proper treatment of CMOS-HD-MEA chips, the management of solutions and brain slices during experimentation, and equipment maintenance.

Author Spotlight: Unraveling Seizure Dynamics and Novel Therapeutics for Status Epilepticus Using CMOS High-Density Microelectrode Array Systems

Here, we outline a protocol for using complementary metal-oxide-semiconductor high-density microelectrode array systems (CMOS-HD-MEAs)&#160;to record seizure-like activity from ex vivo brain slices.

Complementary metal-oxide-semiconductor high-density microelectrode array (CMOS-HD-MEA) systems can record neurophysiological activity from cell cultures ...

Electroencephalographic Recording of Epileptic Seizures in Epilepsy-Induced Rats

Source: Soukupov&#225;, M., et al. <a href="https://app.jove.com/v/58455">Microdialysis of Excitatory Amino Acids During EEG Recordings in Freely Moving Rats.</a> J. Vis. Exp. (2018)
This video demonstrates electroencephalographic (EEG) recording of epileptic seizures in epilepsy-induced rats. A rat with electrodes implanted in the ventral hippocampus is connected to the EEG recording system and placed in a plexiglass chamber. The rat is allowed to move freely while the brain activity is recorded to monitor the occurrence of seizures.

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. ...

JoVE Encyclopedia of Experiments

Neurophysiology

Stimulation and Modulation Techniques

Electroencephalography-Based Recording Technique to Record the Epileptiform Activity

Source: Carvalho, M. et al., <a href="https://app.jove.com/v/61330">A Model of Epileptogenesis in Rhinal Cortex-Hippocampus Organotypic Slice Cultures.</a> J. Vis. Exp. (2021).
This video describes the technique for recording the epileptiform activity of rhinal cortex-hippocampus organotypic slices ex vivo using electrophysiology or EEG set-up. This helps to study the dynamics and progression of epileptogenesis and screen potential therapeutic targets to treat epilepsy.

Source: Carvalho, M. et al., A Model of Epileptogenesis in Rhinal Cortex-Hippocampus Organotypic Slice Cultures. J. Vis. Exp. (2021).
This video describes ...

Biological Techniques

Assay Techniques

Cell-based assays

Monitoring Seizure-Induced Neural Electrical Activity in Brain Slices Using Microelectrode Arrays

Source: Panuccio, G., et al. <a href="https://app.jove.com/v/57548">Recording and modulation of epileptiform activity in rodent brain slices coupled to microelectrode arrays.</a> J. Vis. Exp. (2018)
This protocol involves using a microelectrode array (MEA) to record electrical activity in a brain slice exposed to a seizure-inducing drug. The procedure includes positioning the brain slice in an MEA recording chamber, applying electrical stimulation to induce artificial seizures, and analyzing the resulting signals.

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee.
1. Preparation of the MEA ...

Electrophysiology Techniques

A Micro-drive Array Method for Electrophysiological Recording from Multiple Brain Regions

Source: Shikano, Y., et al. <a href="https://app.jove.com/v/56980">Simultaneous Recordings of Cortical Local Field Potentials, Electrocardiogram, Electromyogram, and Breathing Rhythm from a Freely Moving Rat.</a> J. Vis. Exp. (2018).
The video demonstrates simultaneous electrophysiological recording from multiple brain regions in a rat using an integrative micro-drive array. Recording electrodes are placed in the olfactory bulb, reference electrodes in the frontal cortex, and micro-drive array electrodes in the hippocampus. The rat is allowed to forage freely, and simultaneous recording from all the implanted electrodes is recorded using the array.

Electroencephalography Recording in a Rat Model of Focal Epilepsy

Source: Bae, J. et al., <a href="https://app.jove.com/v/52700">Brain Source Imaging in Preclinical Rat Models of Focal Epilepsy using High-Resolution EEG Recordings.</a>&#160;J. Vis. Exp. (2015)
This video demonstrates a method for high-resolution EEG recording in a rat model of focal epilepsy. It outlines the steps involved in preparing the rat, connecting a multichannel EEG mini-cap to the rat&#39;s scalp, and recording the EEG to identify seizure-related events characteristic of the epilepsy model.

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

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