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. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=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. 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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.

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Research

JoVE Journal

Neuroscience

6.8K ビュー.  Culham Science Centre. 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.

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

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 is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser5. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response1,3,7.
We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.

In vivo Laser Axotomy in C. elegans

A protocol to cut neurons in C. elegans with a MicroPoint pulsed laser is presented. We describe setting up the system, immobilizing worms, and severing labeled neurons. Advantages include a relatively low-cost system and the ability to sever neuronal processes or ablate cells in vivo.

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 characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

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, respectively 4,5,6,7 The GABAA channel is a pentameric GABA-gated chloride channel. The channel subunits are grouped into 8 families (&alpha;1-6, &beta;1-3, &gamma;1-3, &delta;, &#949;, &theta;, &pi; and &rho;). Two alphas, two betas and one 3rd subunit form the functional channel 8. By combining studies of sub-type specific GABA-activated single-channel molecules with studies including all populations of GABAA channels in the neuron it becomes possible to understand the basic mechanism of neuronal inhibition and how it is modulated by pharmacological agents.
We use the patch-clamp technique 9,10 to study the functional properties of the GABAA channels in alive neurons in hippocampal brain slices and record the single-channel and whole-cell currents. We further examine how the channels are affected by different GABA concentrations, other drugs and intra and extracellular factors. For detailed theoretical and practical description of the patch-clamp method please see The Single-Channel Recordings edited by B Sakman and E Neher 10.

We use the patch-clamp technique to measure GABA-activated single-channel currents (GABAA channels, GABAA receptors) and the synaptic and tonic currents they generate in neurons. Activation of the channels decreases neuronal excitability in health and disease 1,2,3,4.

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

The cerebral cortex directs higher cognitive functions. This six layered structure is generated in an inside-first, outside-last manner, in which the first born neurons remain closer to the ventricle while the last born neurons migrate past the first born neurons towards the surface of the brain1. In addition to neuronal migration2, a key process for normal cortical function is the regulation of neuronal morphogenesis3. While neuronal morphogenesis can be studied in vitro in primary cultures, there is much to be learned from how these processes are regulated in tissue environments.
We describe techniques to analyze neuronal migration and/or morphogenesis in organotypic slices of the cerebral cortex4,6. A pSilencer modified vector is used which contains both a U6 promoter that drives the double stranded hairpin RNA and a separate expression cassette that encodes GFP protein driven by a CMV promoter7-9. Our approach allows for the rapid assessment of defects in neurite outgrowth upon specific knockdown of candidate genes and has been successfully used in a screen for regulators of neurite outgrowth8. Because only a subset of cells will express the RNAi constructs, the organotypic slices allow for a mosaic analysis of the potential phenotypes. Moreover, because this analysis is done in a near approximation of the in vivo environment, it provides a low cost and rapid alternative to the generation of transgenic or knockout animals for genes of unknown cortical function. Finally, in comparison with in vivo electroporation technology, the success of ex vivo electroporation experiments is not dependant upon proficient surgery skill development and can be performed with a shorter training time and skill.

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

To conduct a rapid assessment of the function of genes in the development of cerebral cortex, we describe methods involving the ex vivo electroporation of plasmids co-expressing inhibitory RNA (RNAi) and GFP in murine embryonic cortex. This protocol is amenable to the study of various aspects of neurodevelopment such as neurogenesis, neuronal migration and neuronal morphogenesis including dendrite and axon outgrowth.

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 technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

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

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 channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

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

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

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

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 degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

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

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

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

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

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 of surface-expressed receptors is constantly adapting and subject to strict mechanisms of regulation. The paradigmatic example and one of the most studied trafficking events in biology is the regulated control of the synaptic expression of glutamate receptors (GluRs). GluRs mediate the vast majority of excitatory neurotransmission in the central nervous system and control physiological activity-dependent functional and structural changes at the synaptic and neuronal levels (e.g., synaptic plasticity). Modifications in the number, location, and subunit composition of surface expressed GluRs deeply affect neuronal function and, in fact, alterations in these factors are associated with different neuropathies. Presented here is a method to study GluR trafficking in dissociated hippocampal primary neurons. An &#34;antibody-feeding&#34; approach is used to differentially visualize GluR populations expressed at the surface and internal membranes. By labeling surface receptors on live cells and fixing them at different times to allow for receptors endocytosis and/or recycling, these trafficking processes can be evaluated and selectively studied. This is a versatile protocol that can be used in combination with pharmacological approaches or overexpression of altered receptors to gain valuable information about stimuli and molecular mechanisms affecting GluR trafficking. Similarly, it can be easily adapted to study other receptors or surface expressed proteins.

This article presents a method to study glutamate receptor (GluR) trafficking in dissociated primary hippocampal cultures. Using an antibody-feeding approach to label endogenous or overexpressed receptors in combination with pharmacological approaches, this method allows for the identification of molecular mechanisms regulating GluR surface expression by modulating internalization or recycling processes.

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

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...