Name: an alternative approach to study primary events in neurodegeneration using ex vivo rat brain slices
Uploaded: 2018-04-11T15:00:00
Description: Watch this Scientific Journal Video about An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices at JoVE.com

This work was funded by Neuro-Bio Ltd. We would like to thank Dr. Giovanni Ferrati and Dr. Sergio Rotondo (Neuro-Bio) for their comments and advice on the manuscript.

All animal studies have been performed under approved protocols.
NOTE: In this section, the sequence of the main phases performed during the experimental procedure and the suggested time interval is provided (Figure 1). Moreover, a step-by-step description of the protocol is supplemented by an illustrative panel, showing critical actions ranging from brain removal to tissue homogenization after the incubation period (Figure 2). The de...

<ol>
	<li>Braak, H., Braak, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropathological+stageing+of+Alzheimer-related+changes.">Neuropathological stageing of Alzheimer-related changes.</a> Acta Neuropathologica. 82 (4), 239-259 (1991).</li><li>Schliebs, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basal+forebrain+cholinergic+dysfunction+in+Alzheimer's+disease--interrelationship+with+beta-amyloid,+inflammation+and+neurotrophin+signaling.">Basal forebrain cholinergic dysfunction in Alzheimer's disease--interrelationship with beta-amyloid, inflammation and neurotrophin signaling.</a> Neurochemical Research. 30 (6-7), 895-908 (2005).</li><li>Schmitz, T. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basal+forebrain+degeneration+precedes+and+predicts+the+cortical+spread+of+Alzheimer's+pathology.">Basal forebrain degeneration precedes and predicts the cortical spread of Alzheimer's pathology.</a> Nature Communications. 7, 13249 (2016).</li><li>Fjell, A. M., McEvoy, L., Holland, D., Dale, A. M., Walhovd, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=What+is+normal+in+normal+aging?+Effects+of+aging,+amyloid+and+Alzheimer's+disease+on+the+cerebral+cortex+and+the+hippocampus.">What is normal in normal aging? Effects of aging, amyloid and Alzheimer's disease on the cerebral cortex and the hippocampus.</a> Progress in neurobiology. 117, 20-40 (2014).</li><li>Kov&#225;cs, T., Cairns, N. J., Lantos, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Olfactory+centres+in+Alzheimer's+disease:+olfactory+bulb+is+involved+in+early+Braak's+stages.">Olfactory centres in Alzheimer's disease: olfactory bulb is involved in early Braak's stages.</a> Neuroreport. 12 (2), 285-288 (2001).</li><li>Winblad, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defeating+Alzheimer's+disease+and+other+dementias:+a+priority+for+European+science+and+society.">Defeating Alzheimer's disease and other dementias: a priority for European science and society.</a> The Lancet Neurology. 15 (5), 455-532 (2016).</li><li>Herrup, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+case+for+rejecting+the+amyloid+cascade+hypothesis.">The case for rejecting the amyloid cascade hypothesis.</a> Nat Neurosci. 18 (6), 794-799 (2015).</li><li>De Strooper, B., Karran, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cellular+Phase+of+Alzheimer's+Disease.">The Cellular Phase of Alzheimer's Disease.</a> Cell. 164 (4), 603-615 (2016).</li><li>Scheltens, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease.">Alzheimer's disease.</a> Lancet. 388 (10043), 505-517 (2016).</li><li>Arendt, T., Br&#252;ckner, M. K., Lange, M., Bigl, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+acetylcholinesterase+and+butyrylcholinesterase+in+Alzheimer's+disease+resemble+embryonic+development-A+study+of+molecular+forms.">Changes in acetylcholinesterase and butyrylcholinesterase in Alzheimer's disease resemble embryonic development-A study of molecular forms.</a> Neurochemistry International. 21 (3), 381-396 (1992).</li><li>Auld, D. S., Kornecook, T. J., Bastianetto, S., Quirion, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease+and+the+basal+forebrain+cholinergic+system:+relations+to+&#946;-amyloid+peptides,+cognition,+and+treatment+strategies.">Alzheimer's disease and the basal forebrain cholinergic system: relations to &#946;-amyloid peptides, cognition, and treatment strategies.</a> Progress in Neurobiology. 68 (3), 209-245 (2002).</li><li>Arendt, T., Bruckner, M. K., Morawski, M., Jager, C., Gertz, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+neurone+loss+in+Alzheimer's+disease:+cortical+or+subcortical?.">Early neurone loss in Alzheimer's disease: cortical or subcortical?.</a> Acta Neuropathol Commun. 3, 10 (2015).</li><li>Mesulam, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Cholinergic+Lesion+of+Alzheimer's+Disease:+Pivotal+Factor+or+The+Cholinergic+Lesion+of+Alzheimer's+Disease:+Pivotal+Factor+or+Side+Show?.">The Cholinergic Lesion of Alzheimer's Disease: Pivotal Factor or The Cholinergic Lesion of Alzheimer's Disease: Pivotal Factor or Side Show?.</a> Learn Mem. , 43-49 (2004).</li><li>Schliebs, R., Arendt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cholinergic+system+in+aging+and+neuronal+degeneration.">The cholinergic system in aging and neuronal degeneration.</a> Behavioural Brain Research. 221 (2), 555-563 (2011).</li><li>Mesulam, M. M., Mufson, E. J., Wainer, B. H., Levey, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+cholinergic+pathways+in+the+rat:+An+overview+based+on+an+alternative+nomenclature+(Ch1-Ch6).">Central cholinergic pathways in the rat: An overview based on an alternative nomenclature (Ch1-Ch6).</a> Neuroscience. 10 (4), 1185-1201 (1983).</li><li>Mesulam, M., Mufson, E. J., Levey, A. I., Wainer, B. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+innervation+of+cortex+by+the+basal+forebrain:+cytochemistry+and+cortical+connections+of+the+septal+area,+diagonal+band+nuclei,+nucleus+basalis+(substantia+innominata),+and+hypothalamus+in+the+rhesus+monkey.">Cholinergic innervation of cortex by the basal forebrain: cytochemistry and cortical connections of the septal area, diagonal band nuclei, nucleus basalis (substantia innominata), and hypothalamus in the rhesus monkey.</a> J Comp Neurol. 214 (2), 170-197 (1983).</li><li>Greenfield, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovering+and+targeting+the+basic+mechanism+of+neurodegeneration:+The+role+of+peptides+from+the+C-terminus+of+acetylcholinesterase:+Non-hydrolytic+effects+of+ache:+The+actions+of+peptides+derived+from+the+C-terminal+and+their+relevance+to+neurodegenerat.">Discovering and targeting the basic mechanism of neurodegeneration: The role of peptides from the C-terminus of acetylcholinesterase: Non-hydrolytic effects of ache: The actions of peptides derived from the C-terminal and their relevance to neurodegenerat.</a> Chemico-Biological Interactions. 203 (3), 543-546 (2013).</li><li>Garcia-Rat&#233;s, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=(I)+Pharmacological+profiling+of+a+novel+modulator+of+the+&#945;7+nicotinic+receptor:+Blockade+of+a+toxic+acetylcholinesterase-derived+peptide+increased+in+Alzheimer+brains.">(I) Pharmacological profiling of a novel modulator of the &#945;7 nicotinic receptor: Blockade of a toxic acetylcholinesterase-derived peptide increased in Alzheimer brains.</a> Neuropharmacology. 105, 487-499 (2016).</li><li>Eimerl, S., Schramm, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+quantity+of+calcium+that+appears+to+induce+neuronal+death.">The quantity of calcium that appears to induce neuronal death.</a> Journal of neurochemistry. 62 (3), 1223-1226 (1994).</li><li>Greenfield, S., Vaux, 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=Commentary+Parkinson's+Disease,+Alzheimer's+Disease+and+Motor+Neurone+Disease:+Identifying+a+Common+Mechanism.">Commentary Parkinson's Disease, Alzheimer's Disease and Motor Neurone Disease: Identifying a Common Mechanism.</a> Science. 113 (3), 485-492 (2002).</li><li>Badin, A. S., Morrill, P., Devonshire, I. M., Greenfield, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=(II)+Physiological+profiling+of+an+endogenous+peptide+in+the+basal+forebrain:+Age-related+bioactivity+and+blockade+with+a+novel+modulator.">(II) Physiological profiling of an endogenous peptide in the basal forebrain: Age-related bioactivity and blockade with a novel modulator.</a> Neuropharmacology. 105, 47-60 (2016).</li><li>Brai, E., Stuart, S., Badin, A. -. S., Greenfield, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Novel+Ex+Vivo+Model+to+Investigate+the+Underlying+Mechanisms+in+Alzheimer's+Disease.">A Novel Ex Vivo Model to Investigate the Underlying Mechanisms in Alzheimer's Disease.</a> Frontiers in Cellular Neuroscience. 11, 291 (2017).</li><li>Cho, S., Wood, A., Bowlby, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+slices+as+models+for+neurodegenerative+disease+and+screening+platforms+to+identify+novel+therapeutics.">Brain slices as models for neurodegenerative disease and screening platforms to identify novel therapeutics.</a> Current neuropharmacology. 5 (1), 19-33 (2007).</li><li>Humpel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+brain+slice+cultures:+A+review.">Organotypic brain slice cultures: A review.</a> Neuroscience. 305, 86-98 (2015).</li><li>Sakmann, B., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patch+clamp+techniques+for+studying+ionic+channels+in+excitable+membranes.">Patch clamp techniques for studying ionic channels in excitable membranes.</a> Annual review of physiology. 46, 455-472 (1984).</li><li>Jensen, M. S., Lambert, J. D. C., Johansen, F. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophysiological+recordings+from+rat+hippocampus+slices+following+in+vivo+brain+ischemia.">Electrophysiological recordings from rat hippocampus slices following in vivo brain ischemia.</a> Brain Research. 554 (1-2), 166-175 (1991).</li><li>Ferrati, G., Martini, F. J., Maravall, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Presynaptic+Adenosine+Receptor-Mediated+Regulation+of+Diverse+Thalamocortical+Short-Term+Plasticity+in+the+Mouse+Whisker+Pathway.">Presynaptic Adenosine Receptor-Mediated Regulation of Diverse Thalamocortical Short-Term Plasticity in the Mouse Whisker Pathway.</a> Frontiers in Neural Circuits. 10, 1-9 (2016).</li><li>Grinvald, A., Hildesheim, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VSDI:+a+new+era+in+functional+imaging+of+cortical+dynamics.">VSDI: a new era in functional imaging of cortical dynamics.</a> Nature Reviews Neuroscience. 5 (11), 874-885 (2004).</li><li>Badin, A. S., John, E., Susan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+spatio-temporal+bioactivity+of+a+novel+peptide+revealed+by+optical+imaging+in+rat+orbitofrontal+cortex+in+vitro:+Possible+implications+for+neurodegenerative+diseases.">High-resolution spatio-temporal bioactivity of a novel peptide revealed by optical imaging in rat orbitofrontal cortex in vitro: Possible implications for neurodegenerative diseases.</a> Neuropharmacology. 73, 10-18 (2013).</li><li>Greenfield, S. A., Badin, A. S., Ferrati, G., Devonshire, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+imaging+of+the+rat+brain+suggests+a+previously+missing+link+between+top-down+and+bottom-up+nervous+system+function.">Optical imaging of the rat brain suggests a previously missing link between top-down and bottom-up nervous system function.</a> Neurophotonics. 4, 31213 (2017).</li><li>Opitz-Araya, X., Barria, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+hippocampal+slice+cultures.">Organotypic hippocampal slice cultures.</a> Journal of visualized experiments: JoVE. (48), (2011).</li><li>Gong, C. -. X., Lidsky, T., Wegiel, J., Grundke-Iqbal, I., Iqbal, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolically+active+rat+brain+slices+as+a+model+to+study+the+regulation+of+protein+phosphorylation+in+mammalian+brain.">Metabolically active rat brain slices as a model to study the regulation of protein phosphorylation in mammalian brain.</a> Brain Research Protocols. 6 (3), 134-140 (2001).</li></ol>

The principal aspect of this protocol, based on the&#160;well-established ex vivo brain technique, allows to synchronously test&#160;two specular hemislices, obtained from the same anatomical plane, monitoring their response after the application of a specific condition (control or treated); this therefore offers an experimental paradigm as tightly controlled as possible. The possibility of evaluating in a time-, dose-, and site-specific manner different neurochemicals related to neuronal impairment, as seen dur...

AD is a chronic pathology characterized by gradual neurodegenerative impairment affecting different brain areas, such as the entorhinal cortex (EC), BF, hippocampus (HC), and olfactory bulb (OB)1,2,3,4,5. The late stages of AD development lead to a progressive cognitive decline, making this disease the most common form of dementia, approximately accounting for 70% of all cases6. Despite extensive attempts to understand the initial stages causing AD, there is not currently a defined experimental indication elucidating them. In addition, the most popular theory - the &#34;amyloid hypothesis&#34; - is increasingly questioned since it does not provide a complete profile in explaining the AD pathobiology, nor a pharmaceutical target that has proved effective7,8,9.
An alternative theory which is receiving increasing attention suggests that the initial mechanisms occurring during neurodegeneration are related to a neuronal cluster primarily susceptible in AD3,10,11,12,13,14. This heterogeneous cellular hub encompassed within the BF, midbrain, and brainstem, projects to multiple regions, such as the EC, HC, and OB15,16. Despite its diversity in neuronal morphology and neurotransmitter synthesis, this core of cells shares a common feature in expressing AChE, which can also have a non-enzymatic function17,18. This non-classical role as a novel signaling molecule mediates&#160;calcium (Ca2+) flow into neurons which can undergo trophic or toxic events in relation to Ca2+ dose, availability, and neuronal age17,18,19.
During neurodegeneration, the observed cellular loss might be therefore associated to this non-enzymatic function17,18,20, which is attributable to a 30mer peptide (T30) cleaved from the AChE C-terminus20. In line with previous results, carried on cell culture and optical imaging18,21 preparations, we demonstrated, through a novel approach based on ex vivo rat brain slices containing BF structures, that&#160;T30 induced&#160;an AD-like profile22. Specifically, this new methodology offers a more physiological scenario than cell culture since it maintains many of the characteristics of an intact tissue, ranging from anatomical to circuitry preservation, albeit for a time window of hours. We applied this protocol to explore the events taking place during the early phases of neurodegeneration, monitoring the acute response upon T30 application.
Despite the large body of literature on using brain slices to investigate molecular pathways implied in neuronal damage or neurogenesis23,24, this protocol provides for the first time a more immediate and sensitive read out compared to the common use of organotypic slices. However, as is the case for organotypic brain sections, this acute slice procedure can also be adopted for several purposes, such as the evaluation of neuroprotective or neurotoxic molecules, discovery of primary molecular changes occurring in a specific process, immunohistochemical analysis, and pharmacological assays for central nervous system related pathologies.

<table>
	<tbody>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>S7653</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>P9333</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate (NaHCO3)</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>S5761</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Magnesium sulphate heptahydrate (MgSO4 (7H2O))</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>63138</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic (KH2PO4)</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>P5655</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Hepes salt</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>H7006</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Hepes acid</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>H3375</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>G7528</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Calcium chloride dehydrate</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>223506</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>T30 peptide</td>
			<td>Genosphere Biotechnologies, France</td>
			<td></td>
			<td>AChE-derived peptide tested</td>
		</tr>
		<tr>
			<td>Surgical dissecting kit</td>
			<td>World Precision Instruments, USA</td>
			<td>Item #: MOUSEKIT</td>
			<td>Brain removal step</td>
		</tr>
		<tr>
			<td>Surgical blades</td>
			<td>Swann-Morton, UK</td>
			<td>BS 2982</td>
			<td>Brain removal step</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Fisher Scientific, USA</td>
			<td>11566873</td>
			<td>Brain preparation for slicing</td>
		</tr>
		<tr>
			<td>Glue</td>
			<td></td>
			<td></td>
			<td>Brain preparation for slicing</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica, Germany</td>
			<td>VT1000 S</td>
			<td>Slicing</td>
		</tr>
		<tr>
			<td>Brushes</td>
			<td></td>
			<td></td>
			<td>Tissue handling</td>
		</tr>
		<tr>
			<td>Oxygen canister</td>
			<td></td>
			<td></td>
			<td>Sectioning and incubation phase</td>
		</tr>
		<tr>
			<td>1x Phosphate buffer saline (PBS)</td>
			<td>Fisher Scientific, USA</td>
			<td>BP2438-4</td>
			<td>Homogenization step</td>
		</tr>
		<tr>
			<td>Phosphatase inhibitors</td>
			<td>Fisher Scientific, USA</td>
			<td>1284-1650</td>
			<td>Homogenization step</td>
		</tr>
		<tr>
			<td>Protease inhibitors</td>
			<td>Roche complete PIC, USA</td>
			<td>4693116001</td>
			<td>Homogenization step</td>
		</tr>
		<tr>
			<td>Pestles</td>
			<td>Starlab, UK</td>
			<td>I1415-5390</td>
			<td>Homogenization step</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce 660 nm Protein Assay</td>
			<td>Thermo Scientific, USA</td>
			<td>22660</td>
			<td>Protein concentration</td>
		</tr>
	</tbody>
</table>

an alternative approach to study primary events in neurodegeneration using ex vivo rat brain slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

The protocol presented here indicates that the administration of a toxic peptide, T30, modulates in a site-dependent manner the expression of &#945;7-nAChR, p-Tau, and A&#946; in BF-containing sections (Figure 3A). The nicotinic receptor shows a significant increase in the rostral-treated hemislice compared to its control counterpart (slice 1, p = 0.0310) (Figure 3B), while the intermediate slice does not reveal any chan...

Watch this Scientific Journal Video about An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices at JoVE.com

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

The overall goal of this procedure is to simultaneously investigate two specular hemislices obtained from the same anatomical plane and montior the effects following a treatment. This method can help answer crucial questions in the molecular neuroscience field such as the primary events effecting key molecules involved in neurodegenerative processes. The main advantage of this technique is that it can provide and medium carried out of molecular cascades underlying neurodegenerative events.Though this method can provide insight into primary events occurring in neurodegeneration, it can also be applied to investigate animal models of different disorders or other organs and tissues properties. Begin with preparing two different artificial cerebrospinal fluids. One used for the brain slicing, and the other for the incubation step.After preparing the slicing ACSF, mix the solution and keep it on ice. Similarly, prepare the recording ACSF, but keep it at room temperature. Oxygenate both solutions.Next, prepare the instruments for the decapitation and brain removal at the dissection hood. Then, set up the vibratome parameters. Set the slice thickness as needed.Such as three hundred microns. Set the frequency at seven and the speed at six. Then, insert the vibratome chamber and surround it with ice.For the brain sectioning, opening the carbogen valve to oxygenate the slicing ACSF. Select the minimum flow rate of around two milliliters per minute. Now in two 15 milliliters tubes, prepare the tus conditions.Load one tube with 10 millilieters of the recording ACSF as a control. Load the other with the 10 milliliter of recording ACSF enriched with the treatment. In this case, two micromolar of T30.Vortex both tubes and keep them on ice until they are needed. Incise the skin over the skull with a surgical blade. Then using scissors, cut the skull along the midline from the posterior to the bone above the olfactory bulbs.Then, laterally pull on the two sides of the skull using forceps to access the brain. Insert a spatula ventrally to the brain. Gently scoop it out and keep it hydrated in ice cold slicing ACSF for about five minutes.After the rest in cold ACSF, transfer the brain to a filter paper. And after having removed the cerebellum, glue the brain vertically on the vibratome disc. Then transfer it to the sectioning chamber filled with ice cold slicing ACSF.Now start the oxygen flow to the chamber solution. Then adjust the cutting interval and proceed with the brain sectioning. Collect the sections containing the region of interest using standard methods.Now carefully divide each section with small scissors along the midline to obtain two specular hemislices. Use each pair of hemislices for the control and treated condition. After being divided, let the hemisections rest in the vibratome chamber for five to ten minutes.After the rest, gently transfer the hemisections using a glass pipet into a bubbling pot containing recording ACSF at room temperature. Then let the hemisections recover for about 30 minutes before proceeding. During the break, load three glass vials with three milliliters of recording ACSF for the control group and three vials with three milliliters of ACSF enriched with T30 for the treated group.After the 30 minute rest, within five minutes, transfer the sample to the baths. First load the consecutive hemislices to the control solution using a brush. Then, using a different brush, transfer the contralateral hemisections to the treatment solution.Next, seal the vials with plastic stoppers containing 21 gauge needles for the delivery of carbogen to the sample. Deliver oxygen at a minimal flow rate, such as two milliliters per minute to prevent any movement of the brain tissue resulting in possible mechanical damage to the tissue. This completes the setup.Now start a five hour incubation. After the incubation, stop the oxygen flow and uncap the vials. Then gently transfer the hemislices using the appropriate brush to 1.5 milliliter tubes containing 250 microliters of lysis buffer maintained on ice.Next using separate pestles, homogenize each tissue. Complete the transfer and homogenization steps within 10 to 15 minutes for all six samples. Now centrifuge the homogenized samples at 1, 000 times gravity for five minutes at four degrees Celsius.After the spin, transfer the supernatants into new tubes and store the samples at negative 80 degrees Celsius. Administration of a toxic peptide, T30, to the basil fore brain containing hemisections had three site specific effects on the tissue. It modulated the expression of alpha seven nicotinic receptor, the phosphorylation of Tau and the expression of amyloid beta.The nicotinic receptor showed a significant increase in the rostral treated hemislice, while the intermediate slice did not reveal any change between the two conditions. In the posterior section, a significant reduction was present in the T30 exposed portion. Phosphorylated Tau levels were significantly increased in the anterior region.On the other hand, the other two basil fore brain sections do not show any significant difference between the conditions. Amyloid beta was significantly enhanced after T30 exposure in the rostral and intermediate hemisections. However, in the codal slide, there was no measurable effect on amyloid beta expression.Once mastered, this technique can be done within seven hours if properly performed.

Research

JoVE Journal

Neuroscience

In vivo Laser Axotomy in C. elegans

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

GABA-activated Single-channel and Tonic Currents in Rat Brain Slices

The GABAA channels are present in all neurons and are located both at synapses and outside of synapses where they generate phasic and tonic currents, ...

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

Methods for Study of Neuronal Morphogenesis: Ex vivo RNAi Electroporation in Embryonic Murine Cerebral Cortex

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent ...

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

An Antibody Feeding Approach to Study Glutamate Receptor Trafficking in Dissociated Primary Hippocampal Cultures

Cellular responses to external stimuli heavily rely on the set of receptors expressed at the cell surface at a given moment. Accordingly, the population ...