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>

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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

14.5K Views.  Istituto Italiano di Tecnologia. 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.

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

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

Quantifying Subcellular Ubiquitin-proteasome Activity in the Rodent Brain

The ubiquitin-proteasome system is a key regulator of protein degradation and a variety of other cellular processes in eukaryotes. In the brain, increases in ubiquitin-proteasome activity are critical for synaptic plasticity and memory formation and aberrant changes in this system are associated with a variety of neurological, neurodegenerative and psychiatric disorders. One of the issues in studying ubiquitin-proteasome functioning in the brain is that it is present in all cellular compartments, in which the protein targets, functional role and mechanisms of regulation can vary widely. As a result, the ability to directly compare brain ubiquitin protein targeting and proteasome catalytic activity in different subcellular compartments within the same animal is critical for fully understanding how the UPS contributes to synaptic plasticity, memory and disease. The method described here allows collection of nuclear, cytoplasmic and crude synaptic fractions from the same rodent (rat) brain, followed by simultaneous quantification of proteasome catalytic activity (indirectly, providing activity of the proteasome core only) and linkage-specific ubiquitin protein tagging. Thus, the method can be used to directly compare subcellular changes in ubiquitin-proteasome activity in different brain regions in the same animal during synaptic plasticity, memory formation and different disease states. This method can also be used to assess the subcellular distribution and function of other proteins within the same animal.

This protocol is designed to efficiently quantify ubiquitin-proteasome system (UPS) activity in different cellular compartments of the rodent brain. Users are able to examine UPS functioning in nuclear, cytoplasmic and synaptic fractions in the same animal, reducing the amount of time and number of animals needed to perform these complex analyses.

The ubiquitin-proteasome system is a key regulator of protein degradation and a variety of other cellular processes in eukaryotes. In the brain, increases ...

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset (Callithrix jacchus)

Marmosets (Callithrix jacchus) are small non-human primates that are gaining popularity in biomedical and preclinical research, including the neurosciences. Phylogenetically, these animals are much closer to humans than rodents. They also display complex behaviors, including a wide range of vocalizations and social interactions. Here, an effective stereotaxic neurosurgical procedure for implantation of recording electrode arrays in the common marmoset is described. This protocol also details the pre- and postoperative steps of animal care that are required to successfully perform such a surgery. Finally, this protocol shows an example of local field potential and spike activity recordings in a freely behaving marmoset 1 week after the surgical procedure. Overall, this method provides an opportunity to study the brain function in awake and freely behaving marmosets. The same protocol can be readily used by researchers working with other small primates. In addition, it can be easily modified to allow other studies requiring implants, such as stimulating electrodes, microinjections, implantation of optrodes or guide cannulas, or ablation of discrete tissue regions.

Stereotaxic Surgery for Implantation of Microelectrode Arrays in the Common Marmoset Callithrix jacchus

This work presents a protocol to perform a stereotaxic, neurosurgical implantation of&#160;microelectrode arrays in the common marmoset. This method specifically enables electrophysiological recordings in freely behaving animals but can be easily adapted to any other similar neurosurgical intervention in this species (e.g., cannula for drug administration or electrodes for brain stimulation).

Marmosets (Callithrix jacchus) are small non-human primates that are gaining popularity in biomedical and preclinical research, including the ...

Optogenetic Activation of Afferent Pathways in Brain Slices and Modulation of Responses by Volatile Anesthetics

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect distinct cellular and network components of these circuits remains unclear. Ex vivo brain slices provide a means by which investigators may probe discrete components of complex networks and disentangle potential mechanisms underlying the effects of volatile anesthetics on evoked responses. To isolate potential cell type- and pathway-specific drug effects in brain slices, investigators must be able to independently activate afferent fiber pathways, identify non-overlapping populations of cells, and apply volatile anesthetics to the tissue in aqueous solution. In this protocol, methods to measure optogenetically-evoked responses to two independent afferent pathways to neocortex in ex vivo brain slices are described. Extracellular responses are recorded to assay network activity and targeted whole-cell patch clamp recordings are conducted in somatostatin- and parvalbumin-positive interneurons. Delivery of physiologically relevant concentrations of isoflurane via artificial cerebral spinal fluid to modulate cellular and network responses is described.

Ex vivo brain slices can be used to study the effects of volatile anesthetics on evoked responses to afferent inputs. Optogenetics are employed to independently activate thalamocortical and corticocortical afferents to non-primary neocortex, and synaptic and network responses are modulated with isoflurane.

Anesthetics influence consciousness in part via their actions on thalamocortical circuits. However, the extent to which volatile anesthetics affect ...

Construction and Implementation of Carbon Fiber Microelectrode Arrays for Chronic and Acute In Vivo Recordings

Multichannel electrode arrays offer insight into the working brain and serve to elucidate neural processes at the single-cell and circuit levels. Development of these tools is crucial for understanding complex behaviors and cognition and for advancing clinical applications. However, it remains a challenge to densely record from cell populations stably and continuously over long time periods. Many popular electrodes, such as tetrodes and silicon arrays, feature large cross-diameters that produce damage upon insertion and elicit chronic reactive tissue responses associated with neuronal death, hindering the recording of stable, continuous neural activity. In addition, most wire bundles exhibit broad spacing between channels, precluding simultaneous recording from a large number of cells clustered in a small area. The carbon fiber microelectrode arrays described in this protocol offer an accessible solution to these concerns. The study provides a detailed method for fabricating carbon fiber microelectrode arrays that can be used for both acute and chronic recordings in vivo. The physical properties of these electrodes make them ideal for stable and continuous long-term recordings at high cell densities, enabling the researcher to make robust, unambiguous recordings from single units across months.

Construction and Implementation of Carbon Fiber Microelectrode Arrays for Chronic and Acute In Vivo Recordings

This protocol describes a procedure for constructing carbon fiber microelectrode arrays for chronic and acute in vivo electrophysiological recordings in mouse (Mus musculus) and ferret (Mustela putorius furo) from multiple brain regions. Each step, following the purchase of raw carbon fibers to microelectrode array implantation, is described in detail, with emphasis on microelectrode array construction.