eoe sub articles

EoE Sub Articles

All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1. Preparation of high-sucrose slice solution and artificial cerebrospinal fluid (ACSF)
<ol>
	<li>Prior to experimental day
	<ol>
		<li>Prepare 1 L of 10x slice pre-solution with laboratory grade Type 1 water containing (in mM): 25 KCl, 20 CaCl2, 10 MgSO4, 12.5 KH2PO4 (Table 1). In order to ...

obtaining horizontal mouse hippocampal brain slices

Source: Van Hoeymissen, E., et al. <a href="https://app.jove.com/v/61753">Horizontal Hippocampal Slices of the Mouse Brain.</a> J. Vis. Exp. (2020)
This video demonstrates a detailed protocol for preparing horizontal hippocampal brain slices from mice, focusing on preserving the integrity of hippocampal fiber pathways. The process involves carefully bisecting and securing the brain, using a vibratome for slicing, and maintaining optimal tissue conditions with a carbogenated fluid, resulting in hippocampal brain slices ready for further experimentation.

<img alt="Figure 1" src="/files/ftp_upload/22602/22602_figure1.jpg" /> 
Figure 1: Detailed information on the preparation of horizontal hippocampal brain slices. (A) Image of tools required for dissection and slicing of the rodent brain: (a) &#177;2 cm long and &#177;0.5 cm wide straps of filter paper (e.g., grade 413); (b) blade; (c) super glue; (d) pipette tip; (e) specimen plate (comes with vibratome); (f) 35 mm culture dish; (g) fine brush; (h) spatula; (i) curve...

Obtaining Horizontal Mouse Hippocampal Brain Slices

 All procedures involving animal samples have been reviewed and approved by the appropriate animal ethical review committee. 
1. Preparation of ...

Take the mouse brain hemispheres in a high-sucrose solution. Secure the hemispheres with their ventral side facing up on a vibratome specimen plate using an adhesive.
Transfer the specimen plate into the slicing chamber.
Add a semi-frozen high-sucrose solution to maintain the tissue&#39;s physiological conditions and solidify the adhesive.
Supply a carbon dioxide-oxygen mixture to maintain tissue oxygenation.
Using set parameters, obtain the initial slices and remove them. Further, obtain horizontal hippocampal slices.
Carefully transfer these brain slices into a chamber containing ACSF for tissue recovery.
The horizontal hippocampal slices are ready for further experimentation.

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Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Primary Neuronal Culture

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 ...

JoVE Journal

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

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 ...

Obtaining Horizontal Mouse Hippocampal Brain Slices

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