Name: generation and on-demand initiation of acute ictal activity in rodent and human tissue
Uploaded: 2019-01-19T12:30:00
Description: Watch this Scientific Journal Video about Generation and On-Demand Initiation of Acute Ictal Activity in Rodent and Human Tissue at JoVE.com

This work was supported by the Canadian Institutes of Health Research (MOP 119603 to Peter L. Carlen and Taufik A. Valiante), the Ontario Brain Institute (to Taufik A. Valiante), and the Mightex Student Research Grant (to Michael Chang). We would like to thank Liam Long for his assistance in filming the video manuscript. We would like to acknowledge Paria Baharikhoob, Abeeshan Selvabaskaran, and Shadini Dematagoda for their assistance in compiling the figures and tables in this manuscript. Figures 1A, 3A, 4A, and 6A are all original figures made from data published in Chang et al.16.

All research involving patients was performed under a protocol approved by the University Health Network Research Ethics Board in accordance with the Declaration of Helsinki. Procedures involving animals were in accordance with guidelines by the Canadian Council on Animal Care and approved by the Krembil Research Institute Animal Care Committee.
1. Protocol I: Acute In vitro Seizure Model
<ol>
	<li>Preparation of dissection solutions and artificial cerebrospinal fluid</stron...

<ol>
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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview:+definitions+and+classifications+of+seizure+emergencies.">Overview: definitions and classifications of seizure emergencies.</a> Journal of Child Neurology. 22 (5_suppl), 9S-13S (2007).</li><li>L&#246;scher, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Critical+review+of+current+animal+models+of+seizures+and+epilepsy+used+in+the+discovery+and+development+of+new+antiepileptic+drugs.">Critical review of current animal models of seizures and epilepsy used in the discovery and development of new antiepileptic drugs.</a> Seizure. 20 (5), 359-368 (2011).</li><li>Jones, R. S., da Silva, A. B., Whittaker, R. G., Woodhall, G. L., Cunningham, M. 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The brain slices are treated with a proconvulsant drug or an altered ACSF perfusate to increase the neural network&#39;s excitability and promote a precipitation of ictal events (electrographic seizure-like events). For mice, the preferred coronal slices of the somatosensory-motor area should contain the cingulate cortex, area 2 (CG), but not the retrosplenial area (RS); these anatomical markers help identify the range of coronal slices that are best for inducing ictal events. An optional modification for mice tissue is ...

Acute seizure models can successfully reproduce electrographic signatures reminiscent of ictal events observed in the electroencephalogram (EEG) of individuals experiencing a seizure. Researchers use these ictal-like events (herein referred to as &#39;ictal events&#39;) as surrogates for the seizure event1. Clinically, ictal events serve as a reliable proxy for seizure events since seizures are a neurological disorder which originates from the brain. In the epilepsy monitoring unit, neurologists rely upon the detection of ictal events to confirm the brain&#39;s epileptogenic region and isolate it for resection2. In the intensive care unit, physicians monitor ictal activity to assess if any seizure activity persists in sedated patients3. Controlling seizures remains to be a challenging issue for the medical community, as 30% of epilepsy patients are drug resistant to the available medication4,5, and 10% of medical cases involving drug-induced seizures are unresponsive to the standard treatment3. This presents a serious concern for the society, as 10% of the American population is prospected to experience one seizure event in their lifetime and 3% are expected to develop epilepsy6.
Studying seizures in chronic epilepsy models is expensive, laborious, and often take months to prepare7. It is also difficult to perform electrophysiological recordings in freely moving animals. Human clinical trials face similar issues, as well as additional complexities related to patient consent, variability in participants&#39; backgrounds, and the moral and ethical considerations involved8. Acute seizure models, on the other hand, are favorable because they are relatively convenient to prepare, cost-efficient, and capable of generating large volumes of ictal events for study9. Additionally, the tissue is fixed in a stable position, so the conditions are ideal for performing the electrophysiological recordings necessary to study seizure dynamics and the related underlying pathophysiology. Acute seizure models remain favorable over in silico (computer) models because they are based on biological material comprised of the brain&#39;s constituent neuronal network with all its inherent factors and synaptic connectivity, that may not be captured by even the most detailed computer models10. These features make acute seizure models poised to be efficient at screening for potential anti-seizure therapies and making preliminary findings before advancing them for further investigation in chronic epilepsy models and clinical trials.
Typically, acute seizure models are derived from the normal brain tissue that has been subjected to hyper-excitable conditions. To induce clinically relevant ictal events in healthy brain tissue, it is important to understand that the brain functions optimally in a critical state11 where excitation (E) and inhibition (I) are balanced12. A disruption of the E-I balance can lead to the hyper-excitable seizure state in which ictal events precipitate. Accordingly, within this conceptual framework, there are two major strategies to generate ictal events in brain slices (in vitro) or in whole-brain (in vivo) preparations: either decreased inhibition (&#34;disinhibition&#34;) or increased excitation (&#34;non-disinhibition&#34;). However, ictal events are highly ordered and synchronized events that require the influence of GABAergic interneurons to orchestrate the neural network activity13,14. For this reason, non-disinhibition models are the most effective for generating ictal events in isolated neural networks, such as in an in vitro brain slice15, whereas in vitro disinhibition models commonly lead to spiking activity reminiscent of interictal-like spiking. Furthermore, within this conceptual framework, a momentary synchronizing event can also reliably trigger an ictal event16. In fact, an ictal event can be triggered by any minor perturbation applied to the neural system17 when it is at a critical state transition (&#34;bifurcation&#34;) point18. Traditionally, these perturbations were induced by electrical stimulation. The recent development of optogenetics in neuroscience, however, now offers a more elegant strategy to induce critical state transitions16.
The methods described in this paper demonstrate how to generate ictal events on-demand in acute seizure models for both in vitro (step 1 of the Protocol) and in vivo studies (step 2 of the Protocol). They involve the choice of brain region, seizure induction method, study type, and species; however, the focus will be on the recommended choice of an acute 4-AP cortical seizure model because of its versatility in a wide variety of study types. The acute in vitro 4-AP seizure model is based on the standard protocol to prepare high-quality brain slices for electrophysiological recordings and imaging studies19. These protocols have already been used to make in vitro coronal brain slices from the somatosensory-motor cortex of mice16,20 and humans21. Modifications to generate ictal events in these types of brain slices have been previously demonstrated16 and the full details are described in the Protocol below. The acute in vivo 4-AP cortical seizure model is based on the standard protocol to prepare a craniotomy for imaging studies22. The modification is that no (glass slide) window is installed following the craniotomy. Instead, proconvulsant agents (4-AP) are topically applied to the exposed cortex to induce ictal events while the animal is under general anesthesia. To our knowledge, our group was the first to develop this acute in vivo cortical seizure model in mice16,23. The acute in vivo 4-AP cortical seizure model prepared from adult mice was developed to complement the in vitro slice model from juvenile tissue. The replication of findings in the adult in vivo seizure model helps to generalize the findings from slice models by addressing the inherent concerns regarding the non-physiological conditions of a 2D brain slice (versus a 3-D whole-brain structure) and the physiological differences between juvenile and adult tissue.
The method of on-demand ictal event initiation is demonstrated using either puffs of neurotransmitters with a picospritzer or optogenetic strategies. To the best of our knowledge, our group is the first to initiate ictal events in human tissue using neurotransmitters via a picospritzer16. For optogenetic strategies, the C57BL/6 mice strain is the conventional strain used for expressing transgenes. The expression of channelrhodopsin-2 (ChR2) in either GABAergic interneurons or glutamatergic pyramidal cells will provide the optional ability to generate ictal events on-demand with brief light pulses. Suitable optogenetic mice strains include the commercially available C57BL/6 variant that expresses ChR2 in either interneurons, using the mouse vesicular GABA transporter promotor (VGAT)24, or pyramidal cells, using the mouse thymus cell antigen 1 promotor (Thy1)25. These commercially available VGAT-ChR2 and Thy1-ChR2 mice offer the opportunity to activate GABAergic neurons or glutamatergic neurons, respectively, in the neocortex with blue (470 nm) light. The ability to generate ictal events on demand in acute seizure models can offer novel opportunities to study seizure initiation dynamics and efficiently evaluate potential anti-seizure therapies.

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	<tbody>
		<tr>
			<td>Sodium pentobarbital</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through the Toronto Western Hospital&#39;s Suppliers</td>
		</tr>
		<tr>
			<td>1 mm syringe</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>25G 5/8&#8221; sterile needle</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Single edge razor blade (2x)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Instant adhesive glue</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Lens paper</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Glass petri dish (2x)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Splinter forceps (2x)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>PVC handle micro spatula</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Micro spoon with flat end</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Detailing brush 5/0</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purcahsed from a boutique art store</td>
		</tr>
		<tr>
			<td>Wide bore transfer pipette</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Dental Tweezer</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Thermometer (digital)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased on Amazon.ca</td>
		</tr>
		<tr>
			<td>Check carbogen tank (95%O2/5%CO2)&#160;</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through the Toronto Western Hospital&#39;s Suppliers</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica</td>
			<td>N/A</td>
			<td>Purchased through the Toronto Western Hospital&#39;s Suppliers</td>
		</tr>
		<tr>
			<td>brain slice incubation chamber (a.k.a. brain slice keeper)&#160;</td>
			<td>Scientific Systems Design Inc</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride (NaCl)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Dextrose</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Magnesium Sulfate (MgSO4 H2O)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Sodium phosphate monobasic monohydrate (HNaPO4&#183;H2O)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Calcium Chloride (CaCl2&#183;2H2O)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Purchased through UT Med Store</td>
		</tr>
	</tbody>
</table>

generation and on-demand initiation of acute ictal activity in rodent and human tissue

Controlling seizures remains a challenging issue for the medical community. To make progress, researchers need a way to extensively study seizure dynamics and investigate its underlying mechanisms. Acute seizure models are convenient, offer the ability to perform electrophysiological recordings, and can generate a large volume of electrographic seizure-like (ictal) events. The promising findings from acute seizure models can then be advanced to chronic epilepsy models and clinical trials. Thus, studying seizures in acute models that faithfully replicate the electrographic and dynamical signatures of a clinical seizure will be essential for making clinically relevant findings. Studying ictal events in acute seizure models prepared from human tissue is also important for making findings that are clinically relevant. The key focus in this paper is on the cortical 4-AP model due to its versatility in generating ictal events in both in vivo and in vitro studies, as well as in both mouse and human tissue. The methods in this paper will also describe an alternative method of seizure induction using the Zero-Mg2+ model and provide a detailed overview of the advantages and limitations of the epileptiform-like activity generated in the different acute seizure models. Moreover, by taking advantage of commercially available optogenetic mouse strains, a brief (30 ms) light pulse can be used to trigger an ictal event identical to those occurring spontaneously. Similarly, 30 - 100 ms puffs of neurotransmitters (Gamma-Amino Butyric Acid or glutamate) can be applied to the human tissue to trigger ictal events that are identical to those occurring spontaneously. The ability to trigger ictal events on-demand in acute seizure models offers the newfound ability to observe the exact sequence of events that underlie seizure initiation dynamics and efficiently evaluate potential anti-seizure therapies.

The application of 100 &#181;M 4-AP to good-quality (undamaged) 450 &#181;m-sized cortical brain slices from a juvenile VGAT-ChR2 mouse reliably induced recurrent ictal events (&#62; 5 s) within 15 min (Figure 1Ai). The application of 100 &#181;M 4-AP to slices of poor-quality resulted in bursting events or spiking activity (Figure 1Aii). On average, 40% of the slices from each dissected mouse brain successfully generated ictal e...

Watch this Scientific Journal Video about Generation and On-Demand Initiation of Acute Ictal Activity in Rodent and Human Tissue at JoVE.com

Generation and On-Demand Initiation of Acute Ictal Activity in Rodent and Human Tissue

Acute seizure models are important for studying the mechanisms underlying epileptiform events. Furthermore, the ability to generate epileptiform events on-demand provides a highly efficient method to study the exact sequence of events underlying their initiation. Here, we describe the acute 4-aminopyridine cortical seizure models established in mouse and human tissue.

Controlling seizures remains a challenging issue for the medical community. To make progress, researchers need a way to extensively study seizure dynamics ...

This method can help answer key questions in the study of seizure mechanisms, such as, what neural subpopulations are responsible for electrographic seizure onset and termination. The main advantage of this technique, is that it can reproduce electrographic seizure events, on demand, and in vivo and in vitro models that are reminiscent of those observed clinically. This technique can also be used to search for potential anti-seizure therapies, by attempting to trigger electrographic seizure events during the application of different anti-seizure drug candidates.To prepare the cortical slices, glue the preclinical brain tissue onto the Vibratome stage, using instant adhesive glue. Gently place the specimin holder into the buffer tray. Insure that the dorsal portion of the brain is facing the Vibratome's blade.Slice the brain into 450 micrometer thick sections, in the dorsal to ventral direction. Make the first cut in the preclinical animal brain to remove the olfactory bulb. Then, make subsequent cuts until the somatosensory motor area is observed.Use a wide bore pipette. Transfer the targeted coronal slices to a Petrie dish containing cold dissection solution. For the coronal slices, make a transverse cut just below the neo cortical commissure, and then, cut at the midline to separate the hemispheres.Then, use a new razor blade to cut off any excess tissue from the slices. It's critical to perform the brain-slicing procedure efficiently. Every moment the brain tissue is not submerged in ACSF, is detrimental to its quality.Damaged, poor quality brain tissue is less likely to generate electrographic seizure events. Transfer the dorsal portion of the coronal slices, that contain the neo cortex, to a second Petrie dish filled with 35 degree Celsius ACSF, for a moment. Then, promptly transfer the slices to an incubation chamber, containing 35 degrees Celsius carbogenated ACSF.Leave the brain slices slightly submerged in the incubation chamber at 35 degrees Celsius for 30 minutes. Then, remove the incubation chamber from the water bath and allow it to return to room temperature. Wait one hour for the brain slices to recover, before performing electrophysiological recordings.In this procedure, cut out lens paper that is slightly larger than the brain slice. Use a wide-bore pipette or a detailing brush to transfer a brain slice onto the cut out lens paper that is held in place using a dental tweezer. Then, transfer the lens paper with a brain slice to the recording chamber, and secure it into position with a harp screen.Subsequently, profuse the brain slice in the recording chamber with carbogenated ACSF at 35 degrees Celsius at a rate of three milliliters per minute. Use a digital thermometer to ensure the recording chamber is 33 to 36 degrees Celsius. Then, backfill the glass electrodes with 10 microliters of ACSF, using a Hamilton syringe.Under the 20 times stereomicroscope, guide the recording glass electrode into the superficial cortical layer, two, three, using manual manipulators. View the electrical activity of the brain slice with standard software. To induce electrographic seizure-like activities, profuse the brain slice with ACSF containing 4-AP at 100 micromolar.View the electrical activity of the brain slice with standard software. To generate electrographic seizure-like events, using optogenetic strategy on the brain slices from optogenetic mice, use a manual manipulator to position a 1, 000 micron cord diameter optical fiber directly above the recording region. Apply a brief pulse of blue light to initiate an ictal event.A user-friendly MATLAB based program was specifically developed to detect and classify the various types of epileptiform events that occur in the in vitro and in vivo 4-AP seizure models. This detection program is available from the Valiante Labs GitHub repository. The application of 100 micromolar 4-AP to good quality 450 micron-sized cortical brain slices from a juvenile VGAT channelrhodopsin mouse, reliably induced recurrent ictal-like events within 15 minutes.The application of 100 micromolar 4-AP to slices of poor quality, resulted in bursting events or spiking activity. On average, 40%of the slices from each dissected preclinical brain, successfully generated ictal-like events. Moreover, 83%of the dissected mice resulted in at least one brain slice that successfully generated ictal-like events.In brain slices with spontaneously occurring ictal-like events, the application of a brief 30 millisecond light pulse on the brain slice, reliably triggered an ictal-like event that was identical in morphology. The same findings were made in brain slices from Thy one Channelrhodopsin two mice. Thus, regardless of which neuronal subpopulation was activated, any brief synchronizing event in the isolated cortical neural network, led to the onset of an ictal-like event.These ictal-like events were comprised of a sentinel spike, tonic-like firing, clonic-like firing, and burst-like activity toward the end. They were similar in nature to the electrographic signatures associated with clinical seizures. Following this procedure, other types of brain state transitions can be performed to address questions like, what are the neuro biological mechanisms underlying brain state transitions?And how can we regulate these transitions to prevent entry into various pathological brain states?

Research

JoVE Journal

Neuroscience

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; ...

Measuring Circadian and Acute Light Responses in Mice using Wheel Running Activity

Circadian rhythms are physiological functions that cycle over a period of approximately 24 hours (circadian- circa: approximate and diem: day)1, 2.  They ...

Generation of Neural Stem Cells from Discarded Human Fetal Cortical Tissue

Neural stem cells (NSCs) reside along the ventricular zone neuroepithelium during the development of the cortical plate. These early progenitors ...

Imaging Pheromone Sensing in a Mouse Vomeronasal Acute Tissue Slice Preparation

Peter Karlson and Martin L&uuml;scher used the term pheromone for the first time in 19591 to describe chemicals used for intra-species communication. ...

Simultaneous Electroencephalography, Real-time Measurement of Lactate Concentration and Optogenetic Manipulation of Neuronal Activity in the Rodent Cerebral Cortex

Although the brain represents less than 5% of the body  by mass, it utilizes approximately one quarter of the glucose used by the body  at rest1. The ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

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, ...

Experimental Strategies to Bridge Large Tissue Gaps in the Injured Spinal Cord after Acute and Chronic Lesion

After a spinal cord injury (SCI) a scar forms in the lesion core which hinders axonal regeneration. Bridging the site of injury after an insult to the ...

Prolonged Incubation of Acute Neuronal Tissue for Electrophysiology and Calcium-imaging

Acute neuronal tissue preparations, brain slices and retinal wholemount, can usually only be maintained for 6 - 8 h following dissection. This limits the ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...