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 details regarding the materials and instructions to build the apparatus and the subsequent phase for the WB analysis are previously described22.
1. Surgical Tools, Vibratome Settings&#160;and Treatment Preparation
NOTE: Execute the following steps before starting the experimental procedure:
<ol>
	<li>Prepare two different artificial cerebrospinal fluids (aCSFs), one used for the brain slicing and the other for the incubation step as previously described29. Briefly, the working concentrations&#160;(in mmol)&#160;of the two aCSFs are for the &#34;slicing&#34; aCSF: 120 NaCl, 5 KCl, 20 NaHCO3, 2.4 CaCl2, 2 MgSO4, 1.2 KH2PO4, and 10 glucose; 6.7 HEPES salt and 3.3 HEPES acid; pH: 7.1. While for the &#34;recording&#34; aCSF: 124 NaCl, 3.7 KCl, 26 NaHCO3, 2 CaCl2, 1.3 MgSO4, 1.3 KH2PO4, and 10 glucose; pH: 7.1.</li>
	<li>Mix well the solutions&#160;and then put&#160;on ice and oxygenate the slicing aCSF in order to have it cold while removing the brain and the subsequent sectioning. Keep at room temperature (RT) and supply oxygen to the recording aCSF.</li>
	<li>Place the instruments under the dissection hood for the decapitation and brain removal, i.e., guillotine, surgical dissection kit (scissors, forceps, blades), and spatula.</li>
	<li>Set up the vibratome parameters by selecting the slice thickness (300 &#181;m in this preparation) and the frequency at 7 and the speed at 6. Insert the vibratome chamber in its appropriate space and surround it with ice.</li>
	<li>Open the carbogen (95% O2, 5% CO2) valve to oxygenate the &#34;slicing&#34; (aCSF) during the brain sectioning. Select the minimum flow rate, around 2 mL/min.</li>
	<li>Take two 15-mL tubes to prepare the conditions to test. Add in one tube 10 mL of the &#34;recording&#34; aCSF alone (control group) and in the other, 10 mL of the &#34;recording&#34; aCSF enriched with T30 at a concentration of 2 &#181;M (treated group). Vortex both tubes and keep them on ice until their use in the setup preparation phase (section 3).</li>
</ol>
2. Anesthesia, Brain Extraction&#160;and Slicing
NOTE: In this study, 9 wild type P14 male Wistar rats were used. Brain extraction and subsequent slicing constitute a critical part of the protocol since they should be quickly performed (within 10 min) in order to prevent or at least slow down tissue degeneration processes.
<ol>
	<li>Add 1.5 mL of 100% w/w of isoflurane on a cotton layer into the induction chamber, place the animal inside, and close the lid. Wait until the animal is deeply anesthetized, confirming the absence of the pedal reflex, and then decapitate the animal using the guillotine.</li>
	<li>Keep the head of the animal within a pot containing ice-cold slicing aCSF during the brain removal step (Figure 2A - C).</li>
	<li>Incise the skin over the skull with a surgical blade, then cut the skull using scissors following the midline, from the posterior part and proceed anteriorly until reaching the bone above the OBs (Figure 2A).</li>
	<li>Pull laterally the two sides of the skull using a pair of forceps in order to have access to the brain (Figure 2B). Insert a spatula ventrally to the brain, gently scoop it out, and keep it hydrated in ice-cold &#34;slicing&#34; aCSF for approximately 5 min (Figure 2C).</li>
	<li>Dispose the brain on filter paper and cut out the cerebellum using a blade (Figure 2D). Glue the brain vertically on the vibratome disc and insert it inside the sectioning chamber, fill it with ice-cold &#34;slicing&#34; aCSF, and provide oxygen (Figure 2E).</li>
	<li>Adjust the cutting interval, and proceed with the brain sectioning (Figure 2F). Collect the sections containing the region of interest, such as the medial septum (MS), diagonal band of Broca (DBB), and substantia innominata (SI), as previously described22. From top to the bottom, the three slices contain the rostral (R), intermediate (I), and caudal (C) portion of the BF (Figure 2G).</li>
	<li>Divide along the midline each section with small scissors, obtaining two specular hemislices (Figure 2H), used individually as the control and treated condition at the same anatomical plane.</li>
	<li>Keep the hemisections in the vibratome chamber for 5 - 10 min, then transfer them with a glass pipette into a bubbling pot containing recording aCSF. Keep at RT for approximately 30 min to recover.</li>
	<li>Fill, in the meantime, three glass vials with 3 mL of recording aCSF alone (previously prepared in step 1.6) for the control group and three vials with 3 mL of aCSF enriched with T30 (previously prepared in step 1.6) for the treated group.</li>
</ol>
3. Setup Preparation
<ol>
	<li>Transfer the hemisections into their corresponding glass vials (Figure 2I) in order to have three consecutive hemislices (including the rostral, intermediate, and caudal BF region) for the control group (green frame, Figure 2J) and their matching counterparts for the treated group (red frame, Figure 2J).</li>
	<li>Transfer&#160;the brain tissue with two different brushes, one for the control hemislices and another for the treated counterparts, in order to prevent any contamination between conditions.</li>
	<li>Seal each glass vial with the corresponding lid, presenting the oxygenating needle (21G) (Figure 2J). Connect the main tube of the apparatus to the carbogen source (Figure 2K). Deliver oxygen to the hemislices throughout the incubation period with a minimal flow rate of 2 mL/min in order to avoid any movement of the brain tissue and possible mechanical damage during the experiment. Start the 5 h incubation. Execute the steps 3.1 - 3.3 within 5 min. 
	NOTE: Check frequently (every 15 - 20 min) whether the oxygen is supplied into all the vials and that its flow is not moving the tissue from the bottom.</li>
</ol>
4. Sample Homogenization
<ol>
	<li>Stop the oxygen flow after the incubation and remove all the lids from the vials. Transfer the hemislices, using the proper brushes, into 1.5 mL tubes containing 250 &#181;L of lysis buffer, maintaining the tubes on ice (Figure 2L). To make the lysis buffer, mix PBS 1x with phosphatase and protease inhibitors cocktails both diluted at 1:100, and homogenize the tissue with different pestles to prevent&#160;contamination among the samples. Complete&#160;these actions within 10 - 15 min.</li>
	<li>Centrifuge the brain lysate at 1,000 x g for 5 min at 4 &#176;C. Transfer the supernatant into new tubes and keep the samples at -80 &#176;C.</li>
</ol>
5. Reading of the Protein Concentration and Western Blot Analysis
<ol>
	<li>Determine the protein concentration of each sample and subsequently prepare the aliquots for Western blot analysis as previously described22.</li>
	<li>Perform statistical analysis to evaluate the effects of the treatment on protein expression.</li>
</ol>

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The authors declare competing financial interests. Susan A. Greenfield is the founder and president of Neuro-Bio Limited, a privately owned Company, and holds shares in the Company. Emanuele Brai and Antonella Cogoni are employees of Neuro-Bio Ltd.

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...

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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, ...

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Research

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Neuroscience

6.9K Views.  Culham Science Centre. We present a method which can 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.

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

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

Developmental Biology

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid. Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments. The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory. Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds. This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3. Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research. Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity. Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment. Here, we discuss the steps taken during the dissection procedure to minimize these problems. Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments. The possible impact of other methodological factors on synaptic function in these animal models (e.g. recording solution components, stimulation parameters) are also discussed. While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models.

This article outlines procedures for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and age-related neurodegenerative diseases, such as Alzheimer&#x2019;s disease.

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Medicine

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 ...

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

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

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

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

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Preparing Acute Brain Slices from the Dorsal Pole of the Hippocampus from Adult Rodents

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of cellular and synaptic properties. Nevertheless, there is an ever increasing need to perform correlated analyses between different experimental modes in addition to slice electrophysiology, for example: immunohistochemistry, molecular biology, in vivo imaging or electrophysiological recording; to answer evermore complex questions of brain function. However, making meaningful conclusions from these various experimental approaches is not straightforward, as even within relatively well described brain structures, a high degree of sub-regional variation of cellular function exists. Nowhere is this better exemplified than in the CA1 of the hippocampus, which has well-defined dorso-ventral properties, based on cellular and molecular properties. Nevertheless, many published studies examine protein expression patterns or behaviorally correlated in vivo activity in the dorsal extent of the hippocampus; and explain findings mechanistically with cellular electrophysiology from the ventro-medial region. This is further confounded by the fact that many acute slice electrophysiological experiments are performed in juvenile animals, when other experimental modes are performed in more mature animals. To address these issues, this method incorporates transcardial perfusion of mature (&#62;60 day old rodents) with artificial cerebrospinal fluid followed by preparation of modified coronal slices including the septal pole of the dorsal hippocampus to record from CA1 pyramidal cells. This process leads to the generation of healthy acute slices of dorsal hippocampus allowing for slice-based cellular electrophysiological interrogation matched to other measures.

The purpose of this protocol is to describe a method to produce slices of the dorsal hippocampus for electrophysiological examination. This procedure employs perfusion with chilled ACSF prior to slice preparation with a near-coronal slicing angle which allows for preservation of healthy principal neurons.

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of ...

Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell resolution using electrophysiology, microscopy, and pharmacology. However, a major consideration in the design of in vitro experiments is the extent to which different slice preparations recapitulate naturalistic patterns of neural activity as observed in vivo. In the intact brain, the hippocampal network generates highly synchronized population activity reflective of the behavioral state of the animal, as exemplified by the sharp-wave ripple complexes (SWRs) that occur during waking consummatory states or non-REM sleep. SWRs and other forms of network activity can emerge spontaneously in isolated hippocampal slices under appropriate conditions. In order to apply the powerful brain slice toolkit to the investigation of hippocampal network activity, it is necessary to utilize an approach that optimizes tissue health and the preservation of functional connectivity within the hippocampal network. Mice are transcardially perfused with cold sucrose-based artificial cerebrospinal fluid. Horizontal slices containing the hippocampus are cut at a thickness of 450 &#956;m to preserve synaptic connectivity. Slices recover in an interface-style chamber and are transferred to a submerged chamber for recordings. The recording chamber is designed for dual surface superfusion of artificial cerebrospinal fluid at a high flow rate to improve oxygenation of the slice. This protocol yields healthy tissue suitable for the investigation of complex and spontaneous network activity in vitro.

This protocol describes the preparation of horizontal hippocampal-entorhinal cortex (HEC) slices from mice exhibiting spontaneous sharp-wave ripple activity. Slices are incubated in a simplified interface holding chamber and recordings are performed under submerged conditions with fast-flowing artificial cerebrospinal fluid to promote tissue oxygenation and the spontaneous emergence of network-level activity.

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell ...

Neuronal Death Assay to Evaluate Cell Death in Ex Vivo Epilepsy Model

Source: Carvalho, M., et al., <a href="https://app.jove.com/v/61330">A Model of Epileptogenesis in Rhinal Cortex-Hippocampus Organotypic Slice Cultures.</a> J. Vis. Exp. (2021)
This video demonstrates the technique of assessing neuronal death in rhinal cortex-hippocampus organotypic slices. Neuronal death occurs via evolving epileptic seizures &#8212; resembling in vivo epilepsy &#8212; and is detected through dual-staining of the dead cells using propidium iodide and immunofluorescence.

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

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Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

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

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

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

Preparing Acute Brain Slices from the Dorsal Pole of the Hippocampus from Adult Rodents

Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity

Test śmierci neuronalnej w celu oceny śmierci komórki w modelu padaczki ex vivo