This work was supported by the University of Basel, Department of Biomedicine, and the Swiss National Science Foundation (31003A_141007). Markus Saxer provided technical support.

Animal experiments were carried out in accordance with the European Communities Council Directive of 22 September 2010 (2010/63/EU) and were reviewed and permitted by Swiss authorities.
1. Setting up Organotypic Entorhino-hippocampal Slice Cultures
<ol>
	<li>Prepare entorhino-hippocampal organotypic slice cultures (EHOSCs) from postnatal day 4 (P4) mouse pups using the static incubation method4,11. Here, use C57Bl6 and CB6F1 mice.
	<ol>
		<li>Take approximately 30 min per mouse pup for the preparation of slice cultures, i.e., 3 hr for a litter of 6 mouse pups. Carry out all steps under sterile conditions in a laminar flow workbench with sterilized surgical instruments.</li>
		<li>In order to avoid a possible interference from the position of the slice along the septo-temporal axis, use only hippocampal slices of the septal half. If not stated otherwise, carry out all steps at RT.</li>
	</ol>
	</li>
	<li>Cut off the head of a P4 mouse pup with surgical scissors. No anesthesia is required for this step.
	<ol>
		<li>Remove the skull around the brain and identify the fissure between the caudal end of the cerebral hemispheres and the midbrain. Carefully remove the part of the brain rostral to the midbrain from the skull and place it in ice-cold preparation medium consisting of MEM supplemented with a stabilized form of L-glutamine.</li>
	</ol>
	</li>
	<li>Continue the dissection under a stereomicroscope with the brain being immersed in the ice cold preparation medium. Remove remaining meninges from the cortical hemispheres. Identify the hippocampus at the medial caudal surface of the hemisphere and separate it together with the adjacent entorhinal cortex from the remainder of the brain.
	<ol>
		<li>Transfer the tissue to a tissue chopper and cut 400 &#181;m thick slices under aseptic conditions transversal to the septo-temporal axis of the hippocampus.</li>
	</ol>
	</li>
	<li>Place the slices on cell culture inserts (approximately 6 slices per insert) and incubate them in 6-well plates with 1 ml per well of incubation medium (47 ml MEM, 25 ml Eagle Medium, 25 ml horse serum, 1 ml glutamax I, 1 ml of a sterile 10% glucose solution, pH 7.3) in a humidified atmosphere with 5% CO2 at 37 &#176;C. Change the medium the next day and then every other day up to 1 week.</li>
</ol>
2. In Vitro Hypoxia and Oxygen-glucose Deprivation with Reperfusion
<ol>
	<li>See Table 1 for an exact time schedule of the procedure. Culture EHOSCs for 5 days (DIV5) before exposure to hypoxia or OGD. At this time point switch the medium from incubation medium containing 25% horse serum to serum free medium with the following composition: Neurobasal medium with 2% B27 supplement, 1% glutamax and additional 0.1% glucose.</li>
	<li>Prepare the OGD medium as detailed in Reference 7 .The final concentrations of the components will be the following: 0.3 mM CaCl2, 70 mM NaCl, 5.25 mM NaHCO3, 70 mM KCl, 1.25 mM NaH2PO4, 2 mM MgSO4, and 10 mM sucrose at pH 6.8. Add 1 ml of OGD medium to each well of two 6-well tissue culture plates.
	<ol>
		<li>Perfuse the OGD medium with N2 for 1 hr in a hypoxia chamber and then seal and keep O/N in an incubator at 37 &#176;C.</li>
		<li>Use anaerobic strips as hypoxic indicators. Consider the medium to be oxygen free when the color of the strips changed from pink to white.</li>
		<li>For the induction of hypoxia, use serum free medium with glucose instead of OGD medium. Before use, perfuse the medium with N2 in a hypoxia chamber as detailed above for OGD medium.</li>
	</ol>
	</li>
	<li>Before the slices are exposed to OGD, perfuse the OGD medium again with N2 for 30 min. Add 2 &#181;l of a 1 mg/ml propidium iodide (PI) solution per well (for a final concentration of 2 &#181;g/ml) to the slices for 30 min and select only healthy slices for experimentation as indicated by low numbers of PI positive cells.</li>
	<li>Use the schedule of OGD exposure and reperfusion indicated in Figure 1. Deprive slices of either oxygen only (hypoxia) or oxygen and glucose (OGD) for 15 min.
	<ol>
		<li>For induction of hypoxia or OGD, transfer slices into OGD medium and keep for 15 min in the hypoxia chamber. Ensure that all fluids that remain on the sides of the insert are aspirated away when the cultures are switched from regular medium to OGD medium and back.</li>
		<li>After hypoxia or OGD, replace the OGD medium with oxygenated serum free medium and cultures are allowed to recover for 3, 24 or 48 hr in serum free medium in the regular incubator at 37 &#176;C with 5% CO2. Add PI again prior to fixation for the determination of cell death.</li>
	</ol>
	</li>
</ol>
3. Pharmacological Treatments of Entorhino-hippocampal Organotypic Slice Cultures
<ol>
	<li>Culture EHOSCs for 5 days (DIV5) in incubation medium before exposure to pharmacological treatments. Start pharmacological treatments either during OGD or immediately after OGD when slices are transferred to serum free medium.</li>
	<li>Treatments for protection from OGD.
	<ol>
		<li>Add 100 &#181;M 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or 1 &#181;M tetrodotoxin (TTX) to the culture medium during OGD induction (for 15 min) or immediately after OGD induction for 30 min. Then, incubate in normoxic serum free medium for 48 hr before fixation.</li>
	</ol>
	</li>
	<li>Treatments for induction of excitotoxic death.
	<ol>
		<li>After 5 days in incubation medium switch cultures to serum free medium containing 100 &#181;M (RS)-&#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) for 30 min.</li>
		<li>Remove the culture medium containing AMPA by aspiration (take care to remove any residual medium on inserts), then transfer the slices to fresh plates with normal serum free medium and keep them for 48 hr until fixation.</li>
	</ol>
	</li>
</ol>
4. Fixation and Immunostaining of Entorhino-hippocampal Organotypic Slice Cultures
<ol>
	<li>Propidium iodide (PI) staining.
	<ol>
		<li>Add PI for 30 min to the culture medium of living slices at a concentration of 2 &#181;g/ml. View live cultures with an inverted microscope equipped with fluorescence optics and select slices for further treatments. For PI staining in combination with immunohistochemistry, add PI solution 30 min prior to fixation of the cultures.</li>
	</ol>
	</li>
	<li>Fixation of the slices.
	<ol>
		<li>Remove the culture medium from the 6-well plate and add 3 ml of freshly prepared 4% paraformaldehyde (PFA) to each well. Then place the 6-well plate in a refrigerator and fix O/N at 4 &#176;C.</li>
		<li>Remove the PFA solution the following day and wash the slices 3-4 times with phosphate buffered saline (PBS).</li>
	</ol>
	</li>
	<li>Removal of the slices from the cell culture insert and blocking procedure for immunohistochemistry of free-floating sections.
	<ol>
		<li>Carefully detach and remove the slices from the culture inserts using an art paint brush of the size 0 or smaller. Transfer each individual slice into a well of a 96 well plate filled with blocking buffer (PBS with 3% normal goat serum (NGS) and 2% Triton-X100). Keep the slices in the blocking buffer for 2 hr at RT&#160;under constant agitation by an orbital shaker.</li>
	</ol>
	</li>
	<li>Immunostaining of the slice cultures
	<ol>
		<li>For labeling blood vessels use polyclonal anti-laminin at a dilution of 1:200 in PBS containing 1% NGS and 0.5% Triton-X100.</li>
		<li>For labeling neurons use anti-microtubule associated protein 2 (MAP2) at a dilution of 1:500 in PBS containing 1% NGS and 0.5% Triton-X100.</li>
		<li>Incubate the slices with primary antibodies for 48 hr at 4 &#176;C with constant agitation. After the incubation period, wash them in PBS with 0.5% Triton-X100 for 3-4 times.</li>
		<li>Dilute secondary antibodies 1:500 in PBS containing 1% NGS and 0.5% Triton-X100. Incubate the slices for 2 hr at RT in the dark with the Alexa fluorophore conjugated secondary antibodies. Use the appropriate goat anti-rabbit or goat anti-mouse secondary antibody conjugated to Alexa 350 or 488.</li>
		<li>Remove the secondary antibody solution and wash the slices three times with Tris-buffered saline (TBS); mount them on glass slides and coverslip the slides with Mowiol.</li>
	</ol>
	</li>
	<li>Microscopy of the stained slices</li>
	<li>View the stained slices either on a standard microscope with epifluorescence equipment or with a confocal microscope. Take images with an appropriate camera. For some images, invert the fluorescence contrast in order to yield darkly stained vessels on a bright background (Figures 5 and 6).</li>
</ol>
5. Determination of Regional Vessel Density in the Cultures
<ol>
	<li>Quantify the vessel density based on vessel crossings as described in Reference 8.</li>
	<li>Use slices stained for laminin for these measurements. Superimpose recorded images of 10X magnification with a 6 x 6 grid, where each grid is equal to 100 x 100 &#181;m per square, total field of 0.25 mm2.</li>
	<li>Overlay three such grids in the CA1, CA3, DG, and EC region (Figure 2). Count the vessels crossing the lines of the grid.</li>
	<li>For obtaining results with a good statistical relevance, make measurements in at least 3 independent experiments, each including data from 3 mouse pups with 3 slices per mouse pup.</li>
	<li>Determine the average value of vessel crossings from the grids for each region and perform a statistical analysis by ANOVA with Bonferroni correction as post hoc test (p&#60;0.05 defined as significant) using appropriate statistics software.</li>
</ol>

<ol>
	<li>Turner, R. C., Dodson, S. C., Rosen, C. L., Huber, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+science+of+cerebral+ischemia+and+the+quest+for+neuroprotection:+navigating+past+failure+to+future+success.">The science of cerebral ischemia and the quest for neuroprotection: navigating past failure to future success.</a> J Neurosurg. 118, 1072-1085 (2013).</li><li>Bacigaluppi, M., Comi, G., Hermann, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+ischemic+stroke.+Part+two:+modeling+cerebral+ischemia.">Animal models of ischemic stroke. Part two: modeling cerebral ischemia.</a> Open Neurol J. 4, 34-38 (2010).</li><li>Woodhams, P. L., Atkinson, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+entorhino-dentate+projections+in+organotypic+slice+cultures:+mode+of+axonal+regrowth+and+effects+of+growth+factors.">Regeneration of entorhino-dentate projections in organotypic slice cultures: mode of axonal regrowth and effects of growth factors.</a> Exp Neurol. 140, 68-78 (1996).</li><li>Prang, P., Del Turco, D., Kapfhammer, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+entorhinal+fibers+in+mouse+slice+cultures+is+age+dependent+and+can+be+stimulated+by+NT-4,+GDNF,+and+modulators+of+G-proteins+and+protein+kinase.">Regeneration of entorhinal fibers in mouse slice cultures is age dependent and can be stimulated by NT-4, GDNF, and modulators of G-proteins and protein kinase.</a> C. 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Animal experiments were carried out in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and were reviewed and permitted by Swiss authorities. The authors have nothing to disclose.

With the methods presented here, hippocampal organotypic slice cultures can be used as a versatile tool to study plastic changes in neural tissue after injury. While organotypic slice cultures have been used in the past for studying neuronal reactions after ischemia6, 7 the new aspect of the simultaneous study of vascular changes significantly enhances the potential applications of this method. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads t...

The central nervous system is particularly sensitive to a loss or reduction of oxygen and glucose supply by the vasculature. Even a rather short interruption of blood supply to the brain can induce a permanent loss of function of the relevant brain areas leading to the typical stroke syndromes. In addition to the neuronal loss in the primary affected areas, there typically is additional delayed neuronal loss through secondary damage. Unfortunately until now, no neuroprotective treatment for the reduction of secondary neuronal death was available1. Research efforts for studying the mechanisms of secondary damage rely on the use of animal models of cerebral ischemia like middle cerebral artery occlusion and various thrombotic occlusion techniques (for a recent review see2). In parallel, also due to limitations and ethical concerns with the use of animal models, organotypic slice culture of various CNS tissues have been used that allow the study of neuronal reactions to various type of injuries3-5.
For studying the neuronal reaction under conditions that mimic ischemic brain injury, the model system of oxygen glucose deprivation (OGD) has been developed. In this model, slice cultures are temporarily exposed to a medium that lacks glucose and has been equilibrated with nitrogen gas in the absence of oxygen. With such a treatment, it is possible to induce neuronal injury and loss which is rather similar to the one observed after ischemic injury in vivo6, 7. In the hippocampus, such a treatment induces neuronal loss specifically in the CA1, but not in the CA3 area or the dentate gyrus of the hippocampus. In contrast, the study of vascular reactions in slice cultures so far has not been widely undertaken. An obvious reason is the lack of circulation and blood vessel perfusion in the slice culture model. However, we have shown previously that it is possible to maintain blood vessels in CNS slice cultures for several days8, 9.
The overall goal of this method is to not only monitor the fate of neurons after OGD but extend the study to the fate and remodeling of the vasculature which is an important part of the injury response. Until now such studies have required the use of animal experiments (De Jong et al., 1999; Cavaglia et al., 2001). In the protocol presented here, we will detail how such studies can be done in entorhino-hippocampal organotypic slice cultures challenged either with hypoxia or by excitotoxic lesions followed by analysis of both neuronal survival and blood vessel reactions. This protocol is based on a previously published study on this subject10 and can be useful for any laboratory interested in neurovascular interactions in the CNS.

<table border="0" cellpadding="0" cellspacing="0" width="573">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Minimum Essential Medium MEM</td>
			<td style="width:71px;">Gibco</td>
			<td>11012-044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glutamax</td>
			<td style="width:71px;">Gibco</td>
			<td>35050-061</td>
			<td>stabilized form of L-glutamine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Millicell cell culture inserts</td>
			<td style="width:71px;">Millipore</td>
			<td style="width:119px;">PICM03050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Basal medium Eagle&#160;</td>
			<td style="width:71px;">Gibco</td>
			<td>41010-026</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Horse serum</td>
			<td style="width:71px;">Gibco</td>
			<td>26050-088</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Neurobasal medium</td>
			<td style="width:71px;">Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">B27 supplement</td>
			<td style="width:71px;">Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anaerobic strips</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td align="right">59886</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Propidium iodide solution</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td>P4864</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">AMPA</td>
			<td style="width:71px;">R&#38;D systems</td>
			<td>0169-10</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">CNQX</td>
			<td style="width:71px;">R&#38;D systems</td>
			<td style="width:119px;">0190/10</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TTX</td>
			<td style="width:71px;">R&#38;D systems</td>
			<td style="width:119px;">1078/1</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">polyclonal anti-laminin</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td>L9393&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">anti-MAP2</td>
			<td style="width:71px;">Abcam</td>
			<td style="width:119px;">ab11267</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Alexa anti mouse 350</td>
			<td style="width:71px;">Molecular Probes</td>
			<td>A11045</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Alexa anti mouse 488</td>
			<td style="width:71px;">Molecular Probes</td>
			<td>A11001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Alexa anti rabbit 350</td>
			<td style="width:71px;">Molecular Probes</td>
			<td>A11046</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Alexa anti rabbit 488</td>
			<td style="width:71px;">Molecular Probes</td>
			<td>A11008</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Statistics software</td>
			<td style="width:71px;">GraphPad Software</td>
			<td style="width:119px;">GraphPad Prism</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">McIlwain tissue chopper</td>
			<td style="width:71px;">Ted Pella</td>
			<td align="right" style="width:119px;">10180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Hypoxia chamber</td>
			<td>Billups-Rothenberg</td>
			<td style="width:119px;">MIC-101</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Oxygen glucose deprivation and hypoxia induce neuronal death and blood vessel reduction specifically in the hippocampal CA1 region. 
OGD or oxygen-deprivation alone for 15 min induced a strong induction of cell death as seen by propidium iodide staining specifically in the CA1 area of the hippocampus (Figure 3) similar as described previously7. With markers to visualize the network of blood vessels, it was found that blood vessel density and organization appeared similar to the c...

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

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 ...

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JoVE Journal

Neuroscience

9.7K Views.  University of Basel.  بروتوكول للثقافات شريحة عضوي النمط-entorhino الحصين، والذي يسمح استنساخ جوانب كثيرة من إصابات الدماغ الدماغية، ويرد. من خلال دراسة التغيرات في neurovasculature بالإضافة إلى التغييرات في الخلايا العصبية، وهذا البروتوكول هو أداة مرنة لدراسة التغيرات ?...

Video: The Analysis of Neurovascular Remodeling in Entorhino-hippocampal 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. ...

Whole Cell Recording from an Organotypic Slice Preparation of Neocortex

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex. Because of the lack of specific pharmacological agents for these channels, we have taken a genetic approach to manipulating channel expression. We use an organotypic culture preparation (16) in order to maintain cell morphology and the laminar pattern of cortex. We typically isolate acute neocortical slices at postnatal days 8-10 and maintain the slices in culture for 3-7 days. This allows us to study neurons at a similar age to those in our work with acute slices and minimizes the development of exuberant excitatory connections in the slice. We record from visually-identified pyramidal neurons in layers II/III or V using infrared illumination (IR-) and differential interference contrast microscopy (DIC) with whole cell patch clamp in current- or voltage-clamp. We use biolistic (Gene gun) transfection of wild type or mutant potassium channel DNA to manipulate expression of the channels to study their function. The transfected cells are easily identified by epifluorescence microscopy after co-transfection with cDNA for green fluorescent protein (GFP). We compare recordings of transfected cells to adjacent, untransfected neurons in the same layer from the same slice.

This is a protocol to prepare and maintain a neocortical slice preparation in organotypic culture for the purpose of making electrical recordings from pyramidal neurons.

We have been studying the expression and functional roles of voltage-gated potassium channels in pyramidal neurons from rat neocortex.  Because of 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 ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...