The authors would like to acknowledge the Bioimaging Unit of Instituto de Medicina Molecular Jo&#227;o Lobo Antunes, for all the suggestions concerning image acquisition.
This project has received funding from the European Union&#8217;s Horizon 2020 research and innovation programme under grant agreement N&#186; 952455, Funda&#231;&#227;o para a Ci&#234;nciae Tecnologia (FCT) through Project PTDC/MEDFAR/30933/2017, and Faculdade de Medicina da Universidade de Lisboa.

The Portuguese law and European Union guidelines (2010/63/EU) were respected in all procedures regarding the protection of animals for scientific purposes. All methods described here were approved by the iMM&#8217;s Institutional Animal Welfare Body (ORBEA-iMM) and the National competent authority (DGAV &#8211; Dire&#231;&#227;o Geral de Alimenta&#231;&#227;o e Veterin&#225;ria).
1. Preparation of rhinal cortex-hippocampus slices
NOTE: The preparation of rhinal cortex-hippocampus slices uses P6-7 Sprague-Dawley rats.
<ol>
	<li>Culture setup and medium preparation
	<ol>
		<li>On the day before the culture, prepare the required media and place them at 4 &#176;C.</li>
		<li>Prepare dissection medium: 25 mM glucose in Gey&#39;s Balanced Salt Solution (GBSS).</li>
		<li>Prepare culture medium: 50% Opti-MEM, 25% HBSS, 25% Horse Serum (HS), 25 mM glucose, 30 &#181;g/mL Gentamycin.</li>
		<li>Prepare maintenance medium: Neurobasal-A (NBA), 2% B27, 1 mM L-glutamine, 30 &#181;g/mL Gentamycin, HS (15%, 10%, 5% and 0%).</li>
	</ol>
	</li>
	<li>Brain harvesting
	<ol>
		<li>Just before starting the culture, add 1.1 mL of culture medium to each well of the 6-well plate with a P1000 pipette and place it at 37 &#176;C.</li>
		<li>Place all the equipment (dissection microscope, tissue chopper, dissecting lamp, dissecting tools, electrodes, plates, inserts and filter papers) inside the biological safety cabinet and sterilize under UV light for 15 minutes.</li>
		<li>Adjust slice thickness to 350 &#181;m.</li>
		<li>Withdraw the GBSS from the fridge. Add 5 mL of GBSS to six Petri dishes. Six Petri dishes will be required per animal.</li>
		<li>Euthanize the rat pup. Perform decapitation by using a sharp scissor at the base of the brain stem of the animal.</li>
		<li>Wash the animal head three times in cold GBSS and take it inside the safety cabinet.</li>
	</ol>
	</li>
	<li>Tissue isolation and preparation of slices
	<ol>
		<li>Firmly insert sharp forceps into the eye sockets to hold the head.</li>
		<li>Using a thin scissor cut the skin/scalp along the midline starting from the vertebral foramen towards the frontal lobes and put it aside.</li>
		<li>Cut in the same way the skull and along the cerebral transverse fissure (space between brain and cerebellum). With curved long forceps, move it apart.</li>
		<li>Discard the olfactory bulbs with a spatula. Remove the brain from the head and place it in ice-cold GBSS with the dorsal surface faced up (Figure 1A).</li>
		<li>Insert the fine forceps into the cerebellum and go along the midline with the spatula opening each hemisphere very carefully (Figure 1B).</li>
		<li>With short curve forceps, carefully remove the excess tissue that covers the hippocampi, without touching the hippocampal structure. Then with a spatula, cut below each hippocampus (Figure 1C).</li>
		<li>Pick up one hemisphere and place it, with hippocampus facing up, onto a filter paper. Repeat the procedure with the other hemisphere and place it parallel to the first one, in the filter paper. Put the filter paper on the tissue chopper, with the hemispheres perpendicular to the blade, and cut the hemispheres in 350 &#956;m slices (Figure 1D).</li>
		<li>Place the sliced tissue into a Petri dish with cold GBSS (Figure 1E).</li>
		<li>Carefully separate the slices using the round tip electrodes (Figure 1F). Keep only the slices with a structurally intact rhinal cortex and hippocampus. DG and CA areas should be perfectly defined, as well as the entorhinal and perirhinal cortex (Figure 1G).</li>
		<li>Place each slice onto the insert (Figure 1H-I), with a spatula and a round tip electrode. Remove excess dissection medium around each slice with a P20 pipette (Figure 1J). Four rhinal cortex-hippocampus slices can be cultured in a single insert (Figure 1K).</li>
	</ol>
	</li>
	<li>Culture maintenance
	<ol>
		<li>Change the medium every other day.</li>
		<li>Warm up the medium at 37 &#176;C.</li>
		<li>Take the plates from the incubator. Pick up each insert by holding the plastic edge with forceps (Figure 1L).</li>
		<li>Use a free hand to aspirate the medium from the well. Place the insert back into the well and add 1 mL, with a P1000 pipette, of fresh warmed medium (Figure 1M). Repeat for all the inserts. Make sure no air bubbles are trapped between the membrane and the medium.</li>
	</ol>
	</li>
</ol>
NOTE: Epileptic-like slices undergo a gradual and controlled deprivation of serum in the medium. From 9 Days In Vitro (DIV) on, slices are maintained in NBA without HS14.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61330/61330fig01.jpg" /> 
Figure 1: Detailed procedure for the preparation of rhinal cortex-hippocampus organotypic slices.&#160;(A) Remove the brain from the head and place it in ice-cold GBSS with the dorsal surface faced up. (B) Insert the forceps into the cerebellum. Open the brain through the midline and remove the excess tissue over the hippocampus. (C) With a spatula cut below the hippocampus, as indicated by the arrows. (D) Place both hippocampi facing up and parallel to each other onto the filter paper and cut 350 &#181;m slices on the tissue chopper. (E) Place the sliced hippocampus in ice-cold GBSS. (F) Separate the slices with the help of round tipped glass electrodes. (G) Choose only the slices that depict an intact rhinal cortex and hippocampus. (H, I) With the help of a round tipped glass electrode push each slice to the spatula and place it on the insert. (J) Remove the GBSS surrounding the slice. (K) Place four slices per insert. (L) To change the medium, lift the insert and aspirate the medium with a glass pipette. (M) Add fresh medium by placing the pipette between the insert and the walls of the 6-wells plate. Make sure there are no air bubbles beneath the slices. <a href="https://www.jove.com/files/ftp_upload/61330/61330fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Electrophysiological recordings
NOTE: Electrophysiological recordings were performed in rhinal cortex-hippocampus organotypic slices at 7, 14 and 21 DIV in an interface-type chamber. Recordings were obtained with an amplifier, digitized and analyzed with software. All recordings were band-pass filtered (eight-pole Bessel filter at 60 Hz and Gaussian filter at 600 Hz).
<ol>
	<li>Setup preparation
	<ol>
		<li>Prepare 50 mL of NBA medium with 1 mL of B27 and 250 &#956;L of L-Glutamine. Warm up at 37 &#176;C.</li>
		<li>Set the electrophysiology setup in close circuit. Verify if the flow rate is 2 mL/min.</li>
		<li>Open the carbox (5% CO2/95% O2) valve and check the water level in the system.</li>
		<li>Put the filter paper in the interface recording chamber to drain excess medium and the lens cleaning paper beneath the frame to supply medium to the slice.</li>
		<li>Turn on the temperature controller, the amplifiers, and the micromanipulator.</li>
		<li>Let the temperature in the interface chamber stabilize at 37 &#176;C before starting the recordings.</li>
		<li>Prepare artificial cerebrospinal fluid (aCSF: 124 mM NaCl, 3mM KCl, 1.2 mM NaH2PO4, 25 mM NaHCO3, 10 mM glucose, 2 mM CaCl2, 1 mM MgSO4 with pH 7.4) and use it to fill the glass electrode with a syringe. Place it in the receiving electrode.</li>
	</ol>
	</li>
	<li>Recordings of spontaneous activity
	<ol>
		<li>Once the temperature is stable, remove the plate from the incubator and cut one slice from the insert with a highly sharp blade. Place it in a 60 mm plate with a drop of medium. Take it to the interface recording chamber.</li>
		<li>Place the slice in the interface chamber with the hippocampus to the bottom right. Place&#160;the receiving electrode in the CA3 pyramidal cell layer.</li>
		<li>Proceed to the continuous acquisition protocol and record for 30 min.</li>
	</ol>
	</li>
</ol>
3. PI uptake assay
NOTE: Cell death was assessed by monitoring the cellular uptake of the fluorescent dye propidium iodide (PI). PI is a polar compound, which enters cells with damaged cell membranes and interacts with DNA emitting red fluorescence (absorbance 493 nm, emission 630 nm). Since PI is not permeant to live cells, it is used to detect dead cells in a population.
<ol>
	<li>PI incubation
	<ol>
		<li>Prepare, in culture medium, a fresh 1:10 dilution of PI stock.</li>
		<li>For PI uptake assay remove the plate from the incubator and carefully raise the insert. Add 13 &#181;L of PI to the medium, with a P20 pipette, obtaining a final concentration of 2 &#181;M.&#160; Agitate slowly the plate before putting the insert back in place. Make sure there are no bubbles beneath the slices.</li>
		<li>Put the slices back in the 37 &#176;C incubator for 2&#160;h.</li>
		<li>Proceed with the immunohistochemistry protocol, as described in the next section. Cover the plates with aluminium, since PI is light sensitive.</li>
	</ol>
	</li>
</ol>
4. Immunohistochemistry
NOTE: In immunohistochemistry a neuron specific antibody, as well as antibodies able to discriminate resting and reactive phenotypes of microglia and astrocytes, were used to evaluate the extend of neuronal death and gliosis in rhinal cortex-hippocampus epileptic-like organotypic slices.
<ol>
	<li>Tissue fixation
	<ol>
		<li>Remove the plate from the incubator and aspirate the medium. Fix the slices with 4% paraformaldehyde (PFA) for 1 h at RT, by adding 1 mL of PFA beneath and above the slices, with a P1000 pipette.</li>
		<li>Remove the PFA and add 1 mL of PBS. Also add PBS beneath and above the slices.</li>
		<li>Keep the slices at 4 &#176;C, in PBS, until further use. Always put parafilm around the plates to avoid drying.</li>
	</ol>
	</li>
	<li>Immunostaining steps
	<ol>
		<li>Wash twice, 10 min each time, with 1 mL of PBS.</li>
		<li>Prepare permeabilization/blocking solution containing 1% Triton-X100, 10% HS and 10% BSA in PBS. Prepare 5% BSA solution.</li>
		<li>Draw two rectangles with the hydrophobic pen (Figure 2A). Cut the slices from the insert (Figure 2B) with a highly sharp blade. Put two slices per slide (Figure 2C) and add 140 &#181;L of permeabilization/blocking solution on the top of each slice, using a P200 pipette. Incubate for 3 h at RT.</li>
		<li>Dilute the primary antibodies to the working dilution in 5% BSA in PBS. Incubate with the primary antibodies overnight at 4 &#176;C.</li>
		<li>Incubate with the secondary antibodies for 4 h at RT. From this step on, protect the plate from light since fluorophores are being worked with.</li>
		<li>Place a 50 &#956;L drop of Hoechst solution on the top of each slice and incubate for 20 min at RT.</li>
		<li>Wash between incubations. Always wash three times, for 10 min each time, with PBS-T.</li>
		<li>Remove Hoechst and wash as recommended.</li>
		<li>Add 50 &#956;L of mounting medium on the top of each slice. Cover with a glass coverslip and surround with nail polish (Figure 2D).</li>
		<li>Let it dry at RT for 24 h.</li>
		<li>Visualize the immunostaining under a confocal microscope. Keep the stained slices at -20 &#176;C.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61330/61330fig02.jpg" /> 
Figure 2: Specific procedure for the immunohistochemistry assay.&#160;(A) With the hydrophobic pen draw two squares in the slide. (B) Cut the piece of insert that contains the slice. (C) Place each slice in the squares drawn with the hydrophobic pen and start the permeabilization/blocking step. (D) After concluding the protocol, finish by mounting the slices in mounting medium, covering with a glass coverslip and surrounding it with nail polish. <a href="https://www.jove.com/files/ftp_upload/61330/61330fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The authors have nothing to disclose.

Animal models of epilepsy have been crucial for the discovery of many AEDs, however they require many animals and most of them are time-consuming due to the latent period for seizure onset. The low-magnesium induction of epileptiform activity in hippocampal acute slices has also been thoroughly revised in the literature3, but acute slices have a 6-12 h viability making it impossible to assess long-term changes. Organotypic slices can be maintain in culture from days to weeks, allowing to overcome the short viability time of acute slices, and models of epileptogenesis in organotypic hippocampal slices have been proposed3,7,8.
Here we describe the preparation of organotypic slices, comprising the rhinal cortex and the hippocampus. These slices take 15-20 min to prepare per animal, starting from animal sacrifice until placement of slices onto the inserts, and 6-8 slices per hemisphere can be obtained. Extra care must be taken when opening the hemisphere to expose the hippocampus and when removing the tissue from the filter paper after slicing. Excess tissue above the hippocampus can also compromise the slice integrity during slicing.
Rhinal cortex-hippocampus organotypic slices depict an evolving epileptic-like activity resembling in vivo epilepsy. After one week in culture, most slices depict mixed interictal and ictal-like activity, which progresses to solely ictal-like events with time in culture. So far, we have recorded few interictal discharges in slices with 2-3 weeks. In this system, epileptic-like activity appears to develop faster than in organotypic hippocampal slices. This might be attributed to the presence of the rhinal cortex, which preserves most of the functional input to the hippocampus. To fully address this issue, a complete characterization of the epileptic signals displayed by these slices throughout time in culture, such as number and duration of ictal events, together with their amplitude and frequency, is currently being performed.
This system can be maintained in culture for more than three weeks, and mimics many molecular correlates of epilepsy, such as neuronal death, activation of microglia and astrocytes and increased production of pro-inflammatory cytokines14, allowing a long-term characterization of these aspects. It also represents an easy-to-use screening platform, where pharmacological interventions targeting specific cellular pathways can be implemented and potential therapeutic targets can be tested. Undoubtedly, the system herein presented can help to further enlighten the mechanisms of epileptogenesis.

Epilepsy, one of the most prevalent neurologic disorders worldwide, is characterized by the periodic and unpredictable occurrence of synchronized and excessive neuronal activity in the brain. Despite the various antiepileptic drugs (AEDs) available, one-third of patients with epilepsy are refractory to therapy1 and continue to experience seizures and cognitive decline. Furthermore, available AEDs hamper cognition due to their relatively generalized actions upon neuronal activity. Epileptogenesis is hard to study in humans, due to the multiple and heterogeneous epileptogenic injuries, long latent periods lasting months to decades, and the misleading effects of anticonvulsant treatment after the first spontaneous seizure.
The identification of potential therapeutic agents for the treatment of epilepsy has become possible due to animal models of epilepsy: 1) genetic models, which use genetically predisposed animals in which seizures occur spontaneously or in response to a sensory stimulus; 2) models of electrical stimulation-induced seizures; and 3) pharmacological models of seizure induction that use pilocarpine (a muscarinic receptor agonist), kainate (a kainate receptor agonist) or 4-aminopyridine (a potassium channel blocker), among others. These models were crucial in the understanding of the behavioral changes, as well as molecular and cellular mechanisms underlying epilepsy, and they have led to the discovery of many AEDs2.
Ex vivo preparations are also a powerful tool to explore the mechanisms underlying epileptogenesis and ictogenesis. Acute hippocampal slices, which enable electrophysiological studies of living cells over a 6-12 h period, and organotypic hippocampal slices that can be preserved in an incubator over a period of days or weeks have been extensively used in studies of epileptiform activity3.&#160;
Organotypic brain slices are prepared from explanted tissue and represent a physiological three-dimensional model of the brain. These slices preserve the cytoarchitecture of the region of interest and include all brain cells and their intercellular communication4.&#160;&#160;The most used region for long-term organotypic cultures is the hippocampus, as this region is affected by neuronal loss in multiple neurodegenerative conditions. They have been widely used to model brain disorders and are considered excellent tools for assessing a drug&#8217;s neuroprotective and therapeutic potential5,6. Models of epileptogenesis, stroke and A&#946;-induced toxicity were described in hippocampal organotypic slices7,8,9,10. Parkinson&#8217;s disease was explored in ventral mesencephalon and striatum, as well as cortex-corpus callosum-striatum-substantia nigra, organotypic slices11. Organotypic cerebellar slice cultures mimic many aspects of axon myelination and cerebellar functions and are a widespread model for investigating novel therapeutic strategies in multiple sclerosis12.
However, clinical research and animal models of epilepsy have suggested that the rhinal cortex, composed by perirhinal and entorhinal cortices, plays a role in seizure generation13. Thus, a model of epileptogenesis in rhinal cortex-hippocampus organotypic slices was established14. Under a gradual and controlled deprivation of serum, rhinal cortex-hippocampus organotypic slices depict evolving epileptic-like events, unlike analogous slices always kept in a serum-containing medium.
In epilepsy, as in many acute and chronic diseases of the central nervous system, the neurocentric vision fails to elucidate the mechanisms underlying disease onset and progression. Clinical and experimental evidence point to brain inflammation, in which microglia and astrocytes play a relevant role, as one of the key players contributing to the epileptic process. Pharmacological experiments in animal models of epilepsy suggest that antiepileptogenic effects can be achieved by targeting pro-inflammatory pathways, and nowadays neuroinflammation is regarded as a novel option for the development of therapeutic approaches for epilepsy15.
Here, we thoroughly describe the preparation of rhinal cortex-hippocampus organotypic slice cultures, as well as the details for recording spontaneous epileptiform activity from them. We highlight that this system mimics several neuroinflammatory aspects of epilepsy, being thus suitable to explore the role of glial cells and neuroinflammation in this pathology. Furthermore, it represents an easy-to-use platform for the screening of potential therapeutic approaches for epilepsy.

<table>
	<tbody>
		<tr>
			<td>50 mL Centrifuge Tube, Conical Bottom</td>
			<td>Corning</td>
			<td>430829</td>
			<td></td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td>Manuel Vieira, Lda</td>
			<td>UN1170</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Axon Instruments</td>
			<td>Axoclamp 900A</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplifier</td>
			<td>Axon Instruments</td>
			<td>Digidata 1440A</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-C3d (goat)</td>
			<td>R&#38;D Systems</td>
			<td>AF2655</td>
			<td>Dilute at a ratio 1:1000</td>
		</tr>
		<tr>
			<td>Anti-CD68 (mouse)</td>
			<td>Abcam</td>
			<td>ab31630-125ug</td>
			<td>Dilute at a ratio 1:250</td>
		</tr>
		<tr>
			<td>Anti-GFAP (mouse)</td>
			<td>Millipore SAS</td>
			<td>MAB360</td>
			<td>Dilute at a ratio 1:500</td>
		</tr>
		<tr>
			<td>Anti-Iba1 (rabbit)</td>
			<td>Abcam</td>
			<td>ab108539</td>
			<td>Dilute at a ratio 1:600</td>
		</tr>
		<tr>
			<td>Anti-NeuN (rabbit)</td>
			<td>Werfen</td>
			<td>16712943S</td>
			<td>Dilute at a ratio 1:500</td>
		</tr>
		<tr>
			<td>Artificial cerebrospinal fluid (aCSF)</td>
			<td></td>
			<td></td>
			<td>Homemade</td>
		</tr>
		<tr>
			<td>B-27&#8482; Supplement (50X), serum free</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Blades for scalpel handle</td>
			<td>Fine Science Tools</td>
			<td>10011-00</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>NZYTech</td>
			<td>MB04602</td>
			<td>5% BSA is used to dilute the primary antibodies. Add 0.5g BSA in 10 mL PBS.</td>
		</tr>
		<tr>
			<td>Brain/Tissue Slice Chamber System</td>
			<td>Warner Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride dihydrate</td>
			<td>Merck Millipore</td>
			<td>1.02382.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture inserts, 30 mm, hydrophilic PTFE</td>
			<td>Millipore SAS</td>
			<td>PICM03050</td>
			<td></td>
		</tr>
		<tr>
			<td>Cold light source</td>
			<td>SCHOTT</td>
			<td>KL 300 LED</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser microscope</td>
			<td>Zeiss</td>
			<td>LSM 710</td>
			<td></td>
		</tr>
		<tr>
			<td>Conventional incubator</td>
			<td>Thermo Scientific Heraeus</td>
			<td>BB15, Function Line</td>
			<td>Set to 37 &#176;C and 5% CO2</td>
		</tr>
		<tr>
			<td>D(+)-Glucose monohydrate</td>
			<td>Merck Millipore</td>
			<td>1.08342.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-Glucose solution, 45% in water</td>
			<td>Sigma</td>
			<td>G8769</td>
			<td></td>
		</tr>
		<tr>
			<td>di-Sodium hydrogen phosphate dihydrate</td>
			<td>Merck Milipore</td>
			<td>1.06580.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting microscope/magnifier</td>
			<td>MEIJI TECHNO CO. LTD</td>
			<td>122285</td>
			<td></td>
		</tr>
		<tr>
			<td>Donkey anti-goat IgG (H+L) coupled to Alexa Fluor 568</td>
			<td>Invitrogen</td>
			<td>A11057</td>
			<td>Dilute at a ratio 1:200</td>
		</tr>
		<tr>
			<td>Donkey anti-mouse IgG (H+L) coupled to Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A21202</td>
			<td>Dilute at a ratio 1:200</td>
		</tr>
		<tr>
			<td>Donkey anti-mouse IgG (H+L) coupled to Alexa Fluor 568</td>
			<td>Invitrogen</td>
			<td>A10037</td>
			<td>Dilute at a ratio 1:200</td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit IgG (H+L) coupled to Alexa Fluor 488</td>
			<td>Invitrogen</td>
			<td>A21206</td>
			<td>Dilute at a ratio 1:500</td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit IgG (H+L) coupled to Alexa Fluor 568</td>
			<td>Invitrogen</td>
			<td>A10042</td>
			<td>Dilute at a ratio 1:500</td>
		</tr>
		<tr>
			<td>Dumont #5 Fine Forceps Biologie Inox</td>
			<td>Fine Science Tools</td>
			<td>11254-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #5 Forceps Standard Inox</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont #7 Forceps Standard Dumoxel</td>
			<td>Fine Science Tools</td>
			<td>11271-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Dumont Medical #7S Forceps Short Curve Inox</td>
			<td>Fine Science Tools</td>
			<td>11273-22</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamycin stock solution, 50 mg/mL</td>
			<td>Thermo Fisher Scientific</td>
			<td>15750-037</td>
			<td></td>
		</tr>
		<tr>
			<td>Gey&#8217;s Balanced Salt Solution (GBSS)</td>
			<td>Biological Industries</td>
			<td>01-919-1A</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Electrodes</td>
			<td>Science Products</td>
			<td>GB150F-10</td>
			<td>Round tips homemade</td>
		</tr>
		<tr>
			<td>Glass Pasteur pipettes, 230 mm</td>
			<td>VWR International</td>
			<td>612-1702</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution (HBSS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>24020-091</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Invitrogen</td>
			<td>H1399</td>
			<td>Stock solution at 2 mg/mL in PBS</td>
		</tr>
		<tr>
			<td>Horse Serum, Heat Inactivated (HS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>26050-088</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Merck Milipore</td>
			<td>1.09057.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrophobic Pen</td>
			<td>Dako</td>
			<td>S200230-2</td>
			<td></td>
		</tr>
		<tr>
			<td>INCU-Line IL10</td>
			<td>VWR</td>
			<td>390-0384</td>
			<td></td>
		</tr>
		<tr>
			<td>Interface chamber</td>
			<td>Warner Instruments</td>
			<td>BSC-HT Haas Top</td>
			<td></td>
		</tr>
		<tr>
			<td>Iris Spatula Curved</td>
			<td>Fine Science Tools</td>
			<td>10092-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Labculture Class II Biological Safety Cabinet</td>
			<td>HERASafe</td>
			<td>HS 12</td>
			<td></td>
		</tr>
		<tr>
			<td>Lens Cleaning Paper</td>
			<td>TIFFEN</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine solution 200 mM (Q)</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030-024</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate heptahydrate</td>
			<td>Merck Millipore</td>
			<td>1.05886.0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro tube 0.5 mL, PP</td>
			<td>SARSTEDT</td>
			<td>72,699</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro tube 1.5 mL, PP</td>
			<td>SARSTEDT</td>
			<td>72.690.001</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro tube 2.0 mL, PP</td>
			<td>SARSTEDT</td>
			<td>72.691</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulators</td>
			<td>Sutter Instrument</td>
			<td>MP-285</td>
			<td></td>
		</tr>
		<tr>
			<td>Miroscope Cover Glasses, 24 mm x 60 mm</td>
			<td>Marienfeld</td>
			<td>102242</td>
			<td></td>
		</tr>
		<tr>
			<td>Nail polish</td>
			<td>Clich&#233;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal-A Medium (NBA)</td>
			<td>Thermo Fisher Scientific</td>
			<td>10888-022</td>
			<td></td>
		</tr>
		<tr>
			<td>Opti-MEM&#174; I Reduced-Serum Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>31985-047</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde, powder</td>
			<td>VWR Chemicals</td>
			<td>2,87,94,295</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Gilson</td>
			<td>M312</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate saline buffer (PBS)</td>
			<td></td>
			<td></td>
			<td>Homemade. PBS with 0.5% Tween-20 (PBS-T) is used to wash slices during the immunohistochemistry assay.</td>
		</tr>
		<tr>
			<td>Phosphate standard solutions, PO&#8324;3- in water</td>
			<td>BDH ARISTAR</td>
			<td>452232C</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette set</td>
			<td>Gilson</td>
			<td>P2, P10, P20, P100, P200, P1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Platinum 5 blades</td>
			<td>Gillette</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>P5405-250g</td>
			<td></td>
		</tr>
		<tr>
			<td>Propidium iodide (PI)</td>
			<td>Sigma-Aldrich</td>
			<td>P4170-25MG</td>
			<td>Stock solution at 1 mg/mL in water.</td>
		</tr>
		<tr>
			<td>Qualitative Filter Paper, Cellulose, Grade 1, 55 mm</td>
			<td>Whatman</td>
			<td>1001-055</td>
			<td>Medium retention 11&#181;m</td>
		</tr>
		<tr>
			<td>Qualitative Filter Paper, Cellulose, Grade 1, 90 mm</td>
			<td>Whatman</td>
			<td>1001-090</td>
			<td>Medium retention 11&#181;m</td>
		</tr>
		<tr>
			<td>Scalpel handle</td>
			<td>Fine Science Tools</td>
			<td>91003-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Slip Tip Insulin Syringe without Needle 1 mL</td>
			<td>SOL-M</td>
			<td>161000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>VWR Chemicals</td>
			<td>27800.360</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dihydrogen phosphate monohydrate</td>
			<td>Merck Millipore</td>
			<td>1.06346.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydrogen carbonate</td>
			<td>Merck Millipore</td>
			<td>1.06329.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide&#8206;</td>
			<td>Merck Milipore</td>
			<td>535C549998</td>
			<td></td>
		</tr>
		<tr>
			<td>Stimulator</td>
			<td>Astro Med Inc GRASS Product Group</td>
			<td>S48 Stimulator</td>
			<td></td>
		</tr>
		<tr>
			<td>Student Scissors Straight SharpSharp 12cm</td>
			<td>Fine Science Tools</td>
			<td>91402-12</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperFrost Plus&#8482; Adhesion slides</td>
			<td>Thermo Fisher Scientific</td>
			<td>J1800AMNZ</td>
			<td></td>
		</tr>
		<tr>
			<td>TC-Treated Sterile 60 x 15mm Tissue Culture Dish</td>
			<td>Corning</td>
			<td>CORN430166</td>
			<td></td>
		</tr>
		<tr>
			<td>TC-Treated Sterile 6-Wells Plates</td>
			<td>Corning</td>
			<td>CORN3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperatue controller</td>
			<td>MEDICAL SYSTEMS CORP.</td>
			<td>TC-102</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Chopper</td>
			<td>The Mickle Laboratory Engineering CO. LTD.</td>
			<td>MTC/2</td>
			<td>Set to 350 &#956;m</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>BDH</td>
			<td>14630</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma</td>
			<td>P2287</td>
			<td></td>
		</tr>
	</tbody>
</table>

a model of epileptogenesis in rhinal cortex-hippocampus organotypic slice cultures

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s neuroprotective and therapeutic potential. Organotypic slices are prepared from explanted tissue and represent a complex multicellular ex vivo environment. They preserve the three-dimensional cytoarchitecture and local environment of brain cells, maintain the neuronal connectivity and the neuron-glia reciprocal interaction. Hippocampal organotypic slices are considered suitable to explore the basic mechanisms of epileptogenesis, but clinical research and animal models of epilepsy have suggested that the rhinal cortex, composed of perirhinal and entorhinal cortices, play a relevant role in seizure generation.
Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Recordings of spontaneous activity from the CA3 area under perfusion with complete growth medium, at physiological temperature and in the absence of pharmacological manipulations, showed that these slices depict evolving epileptic-like events throughout time in culture. Increased cell death, through propidium iodide uptake assay, and gliosis, assessed with fluorescence-coupled immunohistochemistry, was also observed. The experimental approach presented highlights the value of rhinal cortex-hippocampus organotypic slice cultures as a platform to study the dynamics and progression of epileptogenesis and to screen potential therapeutic targets for this brain pathology.

Based on previous descriptions of epileptic signal analysis in organotypic hippocampal slices, interictal epileptiform discharges are here defined as paroxysmal discharges that are clearly distinguished from background activity, with an abrupt change in polarity and occurring at low frequency (&#60;2 Hz). Paroxysmal discharges lasting more than 10 s and occurring at higher frequency (&#8805;2 Hz) are characterized as ictal epileptiform activity. If an ictal event occurs within 10 s after the previous one, these two events are considered as only one ictal event.
Rhinal cortex-hippocampus organotypic slices at 7 DIV (Figure 3A) depict mixed interictal and ictal-like activity. At 14 DIV (Figure 3B), spontaneous activity is characterized by ictal discharges, which evolve to an overwhelming ictal activity at 21 DIV, with ictal events lasting &#62;1 min (Figure 3C).
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61330/61330fig03.jpg" /> 
Figure 3: Spontaneous epileptiform activity of rhinal cortex-hippocampus organotypic slices.&#160;Representative electrographic seizure-like events, recorded from CA3 area in an interface-type chamber, after (A) 7 DIV, (B) 14 DIV and (C) 21 DIV. Seizure details are shown in lower traces. <a href="https://www.jove.com/files/ftp_upload/61330/61330fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
PI uptake assay followed by immunohistochemistry against the neuronal marker NeuN aimed at identifying neuronal death. PI uptake by granular and pyramidal neurons was observed in 7 DIV slices (arrows in Figure 4A), but the number of PI+ neurons increased at 14 DIV (arrows in Figure 4B), corroborating an increased neuronal death with epileptogenesis progression.
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61330/61330fig04.jpg" /> 
Figure 4: Representative images of NeuN and PI stained rhinal cortex-hippocampus organotypic slices.&#160;Images of NeuN stained mature neurons and PI-positive cells were acquired at (A) 7 DIV and (B) 14 DIV, on a confocal laser microscope with a 20x objective. Magnified images of the dashed areas are shown. Arrows point to death neurons (in orange). Scale-bar, 50 &#956;m. <a href="https://www.jove.com/files/ftp_upload/61330/61330fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
A double staining of Iba1, together with CD68, was used to evaluate&#160;microglia phenotype. Iba1 is a microglia/macrophages marker, while CD68 is a lysosomal protein expressed in high levels by reactive microglia and in low levels by resting microglia. At 7 DIV slices, ramified microglia with a low CD68 expression (arrows in Figure 5A) are more abundant than Iba1+/CD68+ reactive microglia (arrowheads in Figure 5A), whereas at 14 DIV, in all areas of the hippocampus, Iba1+/CD68+ bushy/amoeboid M1 microglia (arrowheads in Figure 5B) exceed microglia with a low CD68 expression (arrows in Figure 5B). At 14 DIV some Iba1+/CD68+ cells with a hyper-ramification appearance can be pinpointed (open arrowheads&#160;in Figure 5B), which might suggest the occurrence of the M2 anti-inflammatory phenotype of microglia. However, this matter requires further study.
Recent studies demonstrated that different initiating CNS injuries can elicit at least two types of reactive astrocytes, A1 and A2, with A1 astrocytes being neurotoxic16. A1 subtype of astrocytes is characterized by an increased expression of Complement C316,17,18. Complement C3, which plays a central role in the activation of the complement system, generates C3b, which is further degraded to iC3b, C3dg and C3d19. Thus, a double staining of GFAP and C3d was employed to assess astrogliosis. At 7 DIV the expression of C3d is barely detectable (Figure 6A), while in 14 DIV slices hypertrophic GFAP+/C3d+ astrocytes can be observed (arrowheads in Figure 6B), suggesting a progressive activation of A1 astrocytes.
Results demonstrate a progressive activation of microglia and astrocytes throughout the course of epileptogenesis, mimicking the events described in patients with epilepsy and in animal models of this pathology.
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/61330/61330fig5v2.jpg" /> 
Figure 5: Representative images of Iba1 and CD68 stained rhinal cortex-hippocampus organotypic slices.&#160;Images of Iba1 and CD68 stained microglia, and Hoechst stained nuclei, were acquired at (A) 7 DIV and (B) 14 DIV, on a confocal laser microscope with a 20x objective. Magnified images of the dashed areas are shown. Arrows point to Iba1+/CD68-&#160;resting microglia, arrowheads indicate Iba1+/CD68+ bushy/amoeboid&#160;microglia and open arrowheads reveal Iba1+/CD68+ hyper-ramified microglia. Scale-bar, 50 &#956;m. <a href="https://www.jove.com/files/ftp_upload/61330/61330fig5v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/61330/61330fig06.jpg" /> 
Figure 6: Representative images of GFAP and C3d stained rhinal cortex-hippocampus organotypic slices.&#160;Images of GFAP and C3d stained astrocytes, and Hoechst stained nuclei, were acquired at (A) 7 DIV and (B) 14 DIV, on a confocal laser microscope with a 20x objective. Magnified images of the dashed areas are shown. Arrowheads point to GFAP+/C3d+ reactive A1 astrocytes (in yellow). Scale-bar, 50 &#956;m. <a href="https://www.jove.com/files/ftp_upload/61330/61330fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about A Model of Epileptogenesis in Rhinal Cortex-Hippocampus Organotypic Slice Cultures at JoVE.com

A Model of Epileptogenesis in Rhinal Cortex-Hippocampus Organotypic Slice Cultures

Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Under a gradual and controlled deprivation of serum, these slices depict evolving epileptic-like events and can be considered an ex vivo model of epileptogenesis. This system represents an excellent tool for monitoring the dynamics of spontaneous activity, as well as for assessing the progression of neuroinflammatory features throughout the course of epileptogenesis.

Organotypic slice cultures have been widely used to model brain disorders and are considered excellent platforms for evaluating a drug&#8217;s ...

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Neuroscience

6.8K Views.  Faculdade de Medicina da Universidade de Lisboa. Here, we describe the preparation of rhinal cortex-hippocampus organotypic slices. Under a gradual and controlled deprivation of serum, these slices depict evolving epileptic-like events and can be considered an ex vivo model of epileptogenesis. This system represents an excellent tool for monitoring the dynamics of spontaneous activity, as well as for assessing the progression of neuroinflammatory features throughout the course of epileptogenesis.

Video: A Model of Epileptogenesis in Rhinal Cortex-Hippocampus Organotypic Slice Cultures

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the anterior hypothalamus. The clock is directly synchronized by light via the retina and optic nerve. Circadian oscillations are generated by interacting negative feedback loops of a number of so called "clock genes" and their protein products, including the Period (Per) genes. The core clock is also dependent on membrane depolarization, calcium and cAMP 1. The SCN shows daily oscillations in clock gene expression, metabolic activity and spontaneous electrical activity. Remarkably, this endogenous cyclic activity persists in adult tissue slices of the SCN 2-4. In this way, the biological clock can easily be studied in vitro, allowing molecular, electrophysiological and metabolic investigations of the pacemaker function.
The SCN is a small, well-defined bilateral structure located right above the optic chiasm 5. In the rat it contains ~8.000 neurons in each nucleus and has dimensions of approximately 947 &mu;m (length, rostrocaudal axis) x 424 &mu;m (width) x 390 &mu;m (height) 6. To dissect out the SCN it is necessary to cut a brain slice at the specific level of the brain where the SCN can be identified. Here, we describe the dissecting and slicing procedure of the SCN, which is similar for mouse and rat brains. Further, we show how to culture the dissected tissue organotypically on a membrane 7, a technique developed for SCN tissue culture by Yamazaki et al. 8. Finally, we demonstrate how transgenic tissue can be used for measuring expression of clock genes/proteins using dynamic luciferase reporter technology, a method that originally was used for circadian measurements by Geusz et al. 9. We here use SCN tissues from the transgenic knock-in PERIOD2::LUCIFERASE mice produced by Yoo et al. 10. The mice contain a fusion protein of PERIOD (PER) 2 and the firefly enzyme LUCIFERASE. When PER2 is translated in the presence of the substrate for luciferase, i.e. luciferin, the PER2 expression can be monitored as bioluminescence when luciferase catalyzes the oxidation of luciferin. The number of emitted photons positively correlates to the amount of produced PER2 protein, and the bioluminescence rhythms match the PER2 protein rhythm in vivo 10. In this way the cyclic variation in PER2 expression can be continuously monitored real time during many days. The protocol we follow for tissue culturing and real-time bioluminescence recording has been thoroughly described by Yamazaki and Takahashi 11.

The procedure of preparing slices containing the adult mouse hypothalamic suprachiasmatic nucleus (SCN), and a rapid way to culture the SCN tissue in organotypic culture condition, are reported. Further, the measurement of oscillatory clock gene protein expression using dynamic luciferase reporter technology is described.

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the ...

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Organotypic Cerebellar Cultures: Apoptotic Challenges and Detection

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. Since then, the technique was developed further and currently there are many different ways to prepare organotypic cultures. The method presented here was adapted from the one described by Stoppini et al. for the preparation of the slices and from Gogolla et al. for the staining procedure 3,4.
A unique feature of this technique is that it allows you to study different parts of the brain such as hippocampus or cerebellum in their original structure, providing a big advantage over dissociated cultures in which all the cellular organization and neuronal networks are disrupted. In the case of the cerebellum it is even more advantageous because it allows the study of Purkinje cells, extremely difficult to obtain as dissociated primary culture. This method can be used to study certain developmental features of the cerebellum in vitro, as well as for electrophysiological and pharmacological experiments in both wild type and mutant mice.
The method described here was designed to study the effect of apoptotic stimuli such as Fas ligand in the developing cerebellum, using TUNEL staining to measure apoptotic cell death. If TUNEL staining is combined with cell type specific markers, such as Calbindin for Purkinje cells, it is possible to evaluate cell death in a cell population specific manner. The Calbindin staining also serves the purpose of evaluating the quality of the cerebellar cultures.

This method describes the generation of organotypic cerebellar cultures and the effect of certain apoptotic stimuli on the viability of different cerebellar cell types.

Organotypic cultures of neuronal tissue were first introduced by Hogue in 1947 1,2 and have constituted a major breakthrough in the field of neuroscience. ...

Preparing Undercut Model of Posttraumatic Epileptogenesis in Rodents

Partially isolated cortex ("undercut") is an animal model of posttraumatic epileptogenesis. The surgical procedure involves cutting through the sensorimotor cortex and the underneath white matter (undercut) so that a specific region of the cerebral cortex is largely isolated from the neighboring cortex and subcortical regions1-3. After a latency of two or more weeks following the surgery, epileptiform discharges can be recorded in brain slices from rodents1; and electrical or behavior seizures can be observed in vivo from other species such as cat and monkey4-6. This well established animal model is efficient to generate and mimics several important characteristics of traumatic brain injury. However, it is technically challenging attempting to make precise cortical lesions in the small rodent brain with a free hand. Based on the procedure initially established in Dr. David Prince's lab at the Stanford University1, here we present an improved technique to perform a surgery for the preparation of this model in mice and rats. We demonstrate how to make a simple surgical device and use it to gain a better control of cutting depth and angle to generate more precise and consistent results. The device is easy to make, and the procedure is quick to learn. The generation of this animal model provides an efficient system for study on the mechanisms of posttraumatic epileptogenesis.

Partially isolated cortex (&#x201C;undercut&#x201D;) is an efficient animal model of posttraumatic epileptogenesis. Here we demonstrate how to make a novel surgical device and use it to make more precise and consistent lesions to generate this model.

Partially isolated cortex ("undercut") is an animal model of posttraumatic epileptogenesis. The surgical procedure involves cutting through the ...

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

The cortex is spontaneously active, even in the absence of any particular input or motor output. During development, this activity is important for the migration and differentiation of cortex cell types and the formation of neuronal connections1. In the mature animal, ongoing activity reflects the past and the present state of an animal into which sensory stimuli are seamlessly integrated to compute future actions. Thus, a clear understanding of the organization of ongoing i.e. spontaneous activity is a prerequisite to understand cortex function. 
Numerous recording techniques revealed that ongoing activity in cortex is comprised of many neurons whose individual activities transiently sum to larger events that can be detected in the local field potential (LFP) with extracellular microelectrodes, or in the electroencephalogram (EEG), the magnetoencephalogram (MEG), and the BOLD signal from functional magnetic resonance imaging (fMRI). The LFP is currently the method of choice when studying neuronal population activity with high temporal and spatial resolution at the mesoscopic scale (several thousands of neurons). At the extracellular microelectrode, locally synchronized activities of spatially neighbored neurons result in rapid deflections in the LFP up to several hundreds of microvolts. When using an array of microelectrodes, the organizations of such deflections can be conveniently monitored in space and time. 
Neuronal avalanches describe the scale-invariant spatiotemporal organization of ongoing neuronal activity in the brain2,3. They are specific to the superficial layers of cortex as established in vitro4,5, in vivo in the anesthetized rat 6, and in the awake monkey7. Importantly, both theoretical and empirical studies2,8-10 suggest that neuronal avalanches indicate an exquisitely balanced critical state dynamics of cortex that optimizes information transfer and information processing.
In order to study the mechanisms of neuronal avalanche development, maintenance, and regulation, in vitro preparations are highly beneficial, as they allow for stable recordings of avalanche activity under precisely controlled conditions. The current protocol describes how to study neuronal avalanches in vitro by taking advantage of superficial layer development in organotypic cortex cultures, i.e. slice cultures, grown on planar, integrated microelectrode arrays (MEA; see also 11-14).

A robust way to study neuronal avalanches, i.e. scale-invariant spatio-temporal activity bursts, indicative of critical state dynamics in cortex. Avalanches emerge spontaneously in developing superficial layers of cultured cortex which allows for long-term measurements of the activity with planar integrated multi-electrode arrays (MEA) under precisely controlled conditions.

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...