The research in this report was funded by a target grant from the NIGMS (1P20GM103653) awarded to DK.

The following protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of the University of Delaware. Male Sprague Dawley rats approximately 55&#8211;75 days old were used for this protocol.
1. Brain Extraction and Tissue Preparation
<ol>
	<li>Anesthetize rat with isoflurane in anesthesia induction chamber until rat no longer exhibits a response to foot pinch.</li>
	<li>Sacrifice rats via rapid decapitation using a guillotine.</li>
	<li>Cut skin on skull posterior to anterior and clear from top of skull.</li>
	<li>Use rongeurs to carefully remove the back part of skull, and then using small dissecting scissors cut down midline of skull, pushing upward against the top of skull at all times to avoid damaging the brain tissue.</li>
	<li>Use rongeurs to peel away right and left half of skull to expose the brain.</li>
	<li>Use a small spatula to scoop under brain and sever nerves, carefully elevate brain and freeze brains in isopentane chilled on dry ice (at least -20 &#176;C). After this, store brains in a -80 &#176;C freezer until slicing.</li>
	<li>Slice brains in regions of interest at 30&#8211;50 &#181;m in a cryostat maintained between -9 &#176;C and -12 &#176;C. Directly mount slices onto glass slides and store in a -80 &#176;C freezer until time of immunocytochemistry assay. 
	NOTE: In this protocol brain regions of interest were the hippocampus and the amygdala.</li>
</ol>
2. Single Immunohistochemical Reaction
NOTE: For double immunohistochemical reaction, the protocol is the same as the single immunohistochemical reaction except this reaction has two primary antibodies of different hosts (e.g., rabbit and mouse) and two secondary antibodies for the corresponding primaries should be from a single host (e.g., goat antirabbit and goat antimouse). The secondary antibodies also have to be from two different spectra that are available in high-resolution scanners. For example, one secondary antibody with an emission spectrum peak at 680 nm and one secondary antibody 800CW (emission spectrum peak at 780 nm).
<ol>
	<li>Remove glass slides from the freezer and allow to equilibrate to room temperature for 30 min.</li>
	<li>Under a fume hood, fix brain tissue in 4% paraformaldehyde in 0.1 M phosphate buffered saline (PBS) for 1&#8722;2 h at room temperature.</li>
	<li>Rinse slides in 0.1 M tris buffered saline (TBS) three times for 10 min each. Permeabilize cell membranes by incubating slides in a mild detergent (e.g., 0.01% detergent) for 30&#8722;60 min.</li>
	<li>Rinse slides in TBS three times for 15 min each.</li>
	<li>Dilute primary antibody for protein of interest in PBS in the correct concentration. For example, to detect the immediate early gene c-Fos, prepare a rabbit primary anti c-Fos antibody in a concentration of 1:500.</li>
	<li>Pipette primary antibody solution directly onto the brain tissue (approximately 200 &#181;L per 3 inch x 1 inch slide).</li>
	<li>Use coverslips to incubate the brain tissue on glass slides in primary antibody dilution for either 1&#8722;2 h at room temperature or overnight (~17 h) at 4 &#176;C.</li>
	<li>Remove coverslips and wash slides in TBS that has a small amount of detergent added to it (e.g., 0.01% detergent, &#34;TBS-T&#34;) four times for 15 min each.</li>
	<li>Use coverslips to incubate brain sections in a secondary antibody at room temperature for 2 h. 
	NOTE: The secondary antibody has to be to the correct dilution and in a diluent containing TBS, detergent, and 1.5% of host serum. For example, a goat secondary antibody in a dilution of 1:2,000 would contain 1.5% goat serum and 0.05% detergent.</li>
	<li>Rinse slides in TBS-T four times for 20 min each, and then in TBS four times for 20 min each.</li>
	<li>Dry slides at room temperature in the dark overnight. When slides are dry, they are ready for imaging.</li>
</ol>
3. Imaging
<ol>
	<li>Place slides onto the near-infrared scanning interface with the tissue facing down. Either image one glass slide or multiple slides at a time using a selection tool.</li>
	<li>Image slides using the highest quality setting with an offset of 0 nm and a resolution of 21 &#181;m. The scanning will typically take 13&#8722;19 h depending on the scanning equipment used.</li>
	<li>Import images into the image analysis software (e.g., ImageStudio) to view and mark for semi-quantitative protein analysis.</li>
</ol>
4. Protein Expression Analysis
<ol>
	<li>Open the image analysis software and select the Work Area into which the image was scanned.</li>
	<li>Open the scanned image in the image analysis software to view the scan, and adjust which wavelengths are viewed, as well as the contrast, brightness, and magnification shown without altering the raw image or the total quantified emission.</li>
	<li>Identify the key regions for quantification and select the Analysis tab along the top of the page, then select Draw Rectangle (or Draw Ellipse/ Draw Freehand) to draw a rectangle over the area that will be quantified.</li>
	<li>To view the size of the rectangle, select Shapes along the bottom left of the screen, then select Columns along bottom right. Add Height and Width columns to identify the shape size. 
	NOTE: It is important to control for shape size when comparing quantification. It is recommended to use identical shapes placed within the desired quantification location, in order to get an accurate sampling of the emissions for that region.</li>
	<li>Then name the shape and repeat. Once all regions are sampled the data available from the columns tab can be aggregated and analyzed.</li>
</ol>

<ol><li>Kandel, E. R. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aKandel%2C+ER+%22Principles+of+Neural+Science%22">Principles of Neural Science</a>. , McGraw Hill. New York. (2013).</li>
<li>Byrne, J. H., Roberts, J. L. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aByrne%2C+JH+%22From+Molecules+to+Networks%3A+An+Introduction+to+Cellular+and+Molecular+Neuroscience%22">From Molecules to Networks: An Introduction to Cellular and Molecular Neuroscience</a>. , Academic Press/Elsevier. (2009).</li>
<li>Salami, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dopamine+D2%2F3+binding+potential+modulates+neural+signatures+of+working+memory+in+a+load-dependent+fashion%5BTitle%5D)+AND+%22Journal+of+Neuroscience%22%5BJournal%5D)">Dopamine D2/3 binding potential modulates neural signatures of working memory in a load-dependent fashion.</a> Journal of Neuroscience. , (2018).</li>
<li>Merali, Z., Khan, S., Michaud, D. S., Shippy, S. A., Anisman, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Does+amygdaloid+corticotropin-releasing+hormone+%28CRH%29+mediate+anxiety-like+behaviors%3F+Dissociation+of+anxiogenic+effects+and+CRH+release%5BTitle%5D)+AND+%22European+Journal+of+Neuroscience%22%5BJournal%5D)">Does amygdaloid corticotropin-releasing hormone (CRH) mediate anxiety-like behaviors? Dissociation of anxiogenic effects and CRH release.</a> European Journal of Neuroscience. 20 (1), 229-239 (2004).</li>
<li>English, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dataset+of+mouse+hippocampus+profiled+by+LC-MS%2FMS+for+label-free+quantitation%5BTitle%5D)+AND+%22Data+in+Brief%22%5BJournal%5D)">Dataset of mouse hippocampus profiled by LC-MS/MS for label-free quantitation.</a> Data in Brief. 7, 341-343 (2016).</li>
<li>David, M. A., Tayebi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detection+of+protein+aggregates+in+brain+and+cerebrospinal+fluid+derived+from+multiple+sclerosis+patients%5BTitle%5D)+AND+%22Frontiers+in+Neurology%22%5BJournal%5D)">Detection of protein aggregates in brain and cerebrospinal fluid derived from multiple sclerosis patients.</a> Frontiers in Neurology. 5, 251(2014).</li>
<li>Oliver, C., Jamur, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Immunocytochemical+methods+and+protocols%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Immunocytochemical methods and protocols.</a> Methods in Molecular Biology. , (2010).</li>
<li>Spitzer, N., Sammons, G. S., Price, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Autofluorescent+cells+in+rat+brain+can+be+convincing+impostors+in+green+fluorescent+reporter+studies%5BTitle%5D)+AND+%22Journal+of+Neuroscience+Methods%22%5BJournal%5D)">Autofluorescent cells in rat brain can be convincing impostors in green fluorescent reporter studies.</a> Journal of Neuroscience Methods. 197 (1), 48-55 (2011).</li>
<li>Knox, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Using+c-Jun+to+identify+fear+extinction+learning-specific+patterns+of+neural+activity+that+are+affected+by+single+prolonged+stress%5BTitle%5D)+AND+%22Behavioural+Brain+Researach%22%5BJournal%5D)">Using c-Jun to identify fear extinction learning-specific patterns of neural activity that are affected by single prolonged stress.</a> Behavioural Brain Researach. 341, 189-197 (2018).</li>
<li>Knox, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neural+circuits+via+which+single+prolonged+stress+exposure+leads+to+fear+extinction+retention+deficits%5BTitle%5D)+AND+%22Learning+%26+Memory%22%5BJournal%5D)">Neural circuits via which single prolonged stress exposure leads to fear extinction retention deficits.</a> Learning & Memory. 23 (12), 689-698 (2016).</li>
<li>Malinow, R., Malenka, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((AMPA+receptor+trafficking+and+synaptic+plasticity%5BTitle%5D)+AND+%22Annual+Review+of+Neuroscience%22%5BJournal%5D)">AMPA receptor trafficking and synaptic plasticity.</a> Annual Review of Neuroscience. 25, 103-126 (2002).</li>
<li>Nabavi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Engineering+a+memory+with+LTD+and+LTP%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Engineering a memory with LTD and LTP.</a> Nature. 511 (7509), 348-352 (2014).</li>
<li>Kimmelmann-Shultz, B., Mohammadmirzaei, N., Della Valle, R., Knox, D. Using high resolution near infrared imaging to measure fear-learning induced changes in AMPA/NMDA ratios throughout the fear circuit. The Annual Delaware Neuroscience Research and Poster Symposium, Newark, DE, , (2018).</li>
<li>Eagle, A. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single+prolonged+stress+enhances+hippocampal+glucocorticoid+receptor+and+phosphorylated+protein+kinase+B+levels%5BTitle%5D)+AND+%22Neuroscience+Research%22%5BJournal%5D)">Single prolonged stress enhances hippocampal glucocorticoid receptor and phosphorylated protein kinase B levels.</a> Neuroscience Research. 75 (2), 130-137 (2013).</li>
</ol>

The authors have nothing to disclose.

The results presented in this article show that near-infrared immunocytochemistry in combination with high-resolution scanning can be used to obtain semi-quantitative measures of protein expression in brain tissue. It can also be used to label two proteins simultaneously in the same brain region. We have previously used near-infrared immunohistochemistry to measure immediate early gene expression in multiple brain regions9,10. Immediate early genes can be used as...

The study of neuroscience concerns an investigation of how cells in the brain mediate specific functions1. These can be cellular in nature such as how glia cells confer immunity in the central nervous system or can involve experiments that aim to explain how the activity of neurons in the dorsal hippocampus leads to spatial navigation. In a broad sense, cellular function is determined by the proteins that are expressed in a cell and the activity of these proteins2. As a result, measuring the expression and/or activity of proteins in brain cells are critical for the study of neuroscience.
A number of techniques are available to measure protein expression in the brain. These include in vivo methods such as positron emission topography for receptor densities3 and micro-dialysis for small peptides4. More commonly, ex vivo methods are used to examine protein function and expression. These include mass spectrometry techniques5, western blot and enzyme-linked immunosorbent assay (ELISA)6, and immunocytochemistry7. Immunocytochemistry is widely used in the field of neuroscience. This technique involves the use of a primary antibody to detect a protein (or antigen) of interest (e.g., c-Fos) and a conjugated secondary antibody to detect the protein-primary antibody complex (Figure 1). To enable detection of the protein-primary antibody-secondary antibody complex, secondary antibodies have oxidizing agents such as horseradish peroxidase (HRP) conjugated to them. This allows for the formation of precipitates in cells that can be detected using light microscopy7. Secondary antibodies can also have chemicals fluoresce conjugated to them (i.e., fluorophores). When stimulated these chemicals emit light, which can be used to detect protein-primary antibody-secondary antibody complexes7. Lastly, sometimes primary antibodies have reducing agents and fluorescence chemicals attached to them directly negating the need for secondary antibodies7 (Figure 1).
Interestingly, many immunocytochemistry methods allow for visualization of proteins in brain cells, but not the ability to quantify the amount of protein in a specific cell or brain region. Using light microscopy to detect precipitates from reduction reactions allows for visualization of neurons and glia, but this method cannot be used to quantify protein expression in cells or in a specific brain region. In theory, fluorescence microscopy can be used for this, because the light emitted from the fluorescent secondary antibody is a measure of the protein-primary antibody-secondary antibody complex. However, autofluorescence in brain tissue can make it difficult to use fluorescence microscopy to quantify protein expression in brain tissue8. As a result, light emitted from fluorescent images of brain tissue is rarely used to quantify protein expression in the brain.
Many of these issues can be addressed using near-infrared immunocytochemistry in conjunction with high-resolution scanning9,10. In this article, we describe how immunocytochemistry coupled with fluorophores in the near-infrared emission spectra can be combined with high resolution scanning (e.g., 10&#8211;21 &#181;m) to obtain sharp images that allow for semi quantification of protein in distinct brain regions.

<table><tbody><tr><td>Brain Extraction</td><td></td><td></td><td></td></tr><tr><td>Anesthesia Induction Chamber</td><td>Kent Scientific</td><td>VetFlo-0530SM</td><td></td></tr><tr><td>Kleine Guillotine</td><td>Harvard Apparatus</td><td>73-1920</td><td></td></tr><tr><td>Friedman Rongeur</td><td>Fine Science Tools</td><td>16000-14</td><td>used to remove back of skull</td></tr><tr><td>Delicate Dissecting Scissors</td><td>Fischer Scientific</td><td>08-951-5</td><td>used to cut upward along midline of skull</td></tr><tr><td>Micro Spatula</td><td>Fischer Scientific</td><td>21-401-5</td><td>used to scoop out brain</td></tr><tr><td>Glass Microscope Slides</td><td>Fischer Scientific</td><td>12-549-6</td><td></td></tr><tr><td>Immunohistochemical Reaction</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;Triton X-100</td><td></td><td></td><td>Used as a mild detergent to permeabilize cells after fixing in Paraformaldehyde, also used as mild detergent in combination with host serum and secondary antibody&amp;nbsp;</td></tr><tr><td>Tween-20</td><td></td><td></td><td>Used as a small amount of detergent added to TBS&amp;nbsp; to procuce TBS-T after coverslipping slides with primary antibody</td></tr><tr><td>Licor Odyssey scanner</td><td>Licor Biotechnology Inc.</td><td></td><td></td></tr><tr><td>Image Studio</td><td>Licor Biotechnology Inc.</td><td></td><td></td></tr></tbody></table>

using near-infrared fluorescence and high-resolution scanning to measure protein expression in the rodent brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Prior to using high-resolution scanning for immunohistochemistry, one should verify that the protocol works. This can be accomplished using a validation assay where brain sections from the same animal are incubated with primary and secondary antibodies, secondary antibody alone, or neither primary nor secondary antibody. Results for such a validation assay are shown in Figure 2. In this reaction we were detecting the immediate early gene c-Jun in the dorsal h...

Watch this Scientific Journal Video about Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain at JoVE.com

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...

using-near-infrared-fluorescence-high-resolution-scanning-to-measure

Research

JoVE Journal

Neuroscience

5.5K Views.  University of Delaware. Dit protocol is belangrijk omdat het het mogelijk maakt voor kwantificering van twee eiwitten in hetzelfde hersengebied. Dit stelt de onderzoeker in staat om te kijken naar de activiteit in moleculaire signalering trajecten in hersengebieden door het testen van pan en fosfoproteïnen. Het kan ook worden gebruikt om eiwitten die markers van verschillende cognitieve verschijnselen te testen.We gebruiken een relatief eenvoudige techniek, zoals immunohistochemie, om vragen te beantwoorden ...

Video: Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

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 ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Medicine

Metabolomic Analysis of Rat Brain by High Resolution Nuclear Magnetic Resonance Spectroscopy of Tissue Extracts

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics and proteomics have been complemented by comprehensive quantitative analysis of the metabolite pool present in biological systems. This strategy, termed metabolomics, strives to provide a global characterization of the small-molecule complement involved in metabolism. While the genome and the proteome define the tasks cells can perform, the metabolome is part of the actual phenotype. Among the methods currently used in metabolomics, spectroscopic techniques are of special interest because they allow one to simultaneously analyze a large number of metabolites without prior selection for specific biochemical pathways, thus enabling a broad unbiased approach. Here, an optimized experimental protocol for metabolomic analysis by high-resolution NMR spectroscopy is presented, which is the method of choice for efficient quantification of tissue metabolites. Important strengths of this method are (i) the use of crude extracts, without the need to purify the sample and/or separate metabolites; (ii) the intrinsically quantitative nature of NMR, permitting quantitation of all metabolites represented by an NMR spectrum with one reference compound only; and (iii) the nondestructive nature of NMR enabling repeated use of the same sample for multiple measurements. The dynamic range of metabolite concentrations that can be covered is considerable due to the linear response of NMR signals, although metabolites occurring at extremely low concentrations may be difficult to detect. For the least abundant compounds, the highly sensitive mass spectrometry method may be advantageous although this technique requires more intricate sample preparation and quantification procedures than NMR spectroscopy. We present here an NMR protocol adjusted to rat brain analysis; however, the same protocol can be applied to other tissues with minor modifications.

The neurochemistry of mammalian brain is changed in many neurological and systemic diseases. Characteristic profiles of cerebral metabolites can be efficiently obtained based on crude extracts of brain tissue. To this end, high-resolution NMR spectroscopy is employed, enabling detailed quantitative analysis of metabolite concentrations (metabolomics).

Studies of gene expression on the RNA and protein levels have long been used to explore biological processes underlying disease. More recently, genomics ...

Using Fluorescence Activated Cell Sorting to Examine Cell-Type-Specific Gene Expression in Rat Brain Tissue

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant cell type in the brain, neurons are the most widely studied of these cell types given their direct role in impacting behaviors. Other cell types in the brain also impact neuronal function and behavior via the signaling molecules they produce. Neuroscientists must understand the interactions between the cell types in the brain to better understand how these interactions impact neural function and disease. To date, the most common method of analyzing protein or gene expression utilizes the homogenization of whole tissue samples, usually with blood, and without regard for cell type. This approach is an informative approach for examining general changes in gene or protein expression that may influence neural function and behavior; however, this method of analysis does not lend itself to a greater understanding of cell-type-specific gene expression and the effect of cell-to-cell communication on neural function. Analysis of behavioral epigenetics has been an area of growing focus which examines how modifications of the deoxyribonucleic acid (DNA) structure impact long-term gene expression and behavior; however, this information may only be relevant if analyzed in a cell-type-specific manner given the differential lineage and thus epigenetic markers that may be present on certain genes of individual neural cell types. The Fluorescence Activated Cell Sorting (FACS) technique described below provides a simple and effective way to isolate individual neural cells for the subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA. This technique can also be modified to isolate more specific neural cell types in the brain for subsequent cell-type-specific analysis.

The goal of this protocol is to use the fluorescence activated cell sorting (FACS) technique to sort specific types of neural cells for subsequent analysis of cell-type-specific gene expression, epigenetic markers, and or protein expression.

The brain is comprised of four primary cell types including neurons, astrocytes, microglia and oligodendrocytes. Though they are not the most abundant ...

Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters (CNiFERs) for Optical Detection of Neurotransmitters In Vivo

Cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) provide a new tool for neuroscientists to optically detect the release of neurotransmitters in the brain in vivo. A specific CNiFER is created from a human embryonic kidney cell that stably expresses a specific G protein-coupled receptor, which couples to Gq/11 G proteins, and a FRET-based Ca2+-detector, TN-XXL. Activation of the receptor leads to an increase in the FRET signal. CNiFERs have nM sensitivity and a temporal response of seconds because a CNiFER clone utilizes the native receptor for a particular neurotransmitter, e.g., D2R for dopamine. CNiFERs are directly implanted into the brain, enabling them to sense neurotransmitter release with a spatial resolution of less than one hundred &#181;m, making them ideal to measure volume transmission in vivo. CNiFERs can also be used to screen other drugs for potential cross-reactivity in vivo. We recently expanded the family of CNiFERs to include GPCRs that couple to Gi/o G proteins. CNiFERs are available for detecting acetylcholine (ACh), dopamine (DA) and norepinephrine (NE). Given that any GPCR can be used to create a novel CNiFER and that there are approximately 800 GPCRs in the human genome, we describe here the general procedure to design, realize, and test any type of CNiFER.

Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters CNiFERs for Optical Detection of Neurotransmitters In Vivo

We present a protocol to create cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) for the optical detection of volumetric neurotransmitter release.

Cell-based neurotransmitter fluorescent engineered reporters (CNiFERs) provide a new tool for neuroscientists to optically detect the release of ...

Fluorescence Activated Cell Sorting (FACS) and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific sets of sparsely distributed activated neurons called neuronal ensembles that mediate learned associations. Fluorescence Activated Cell Sorting (FACS) has recently been optimized for adult rat brain tissue and allowed isolation of activated neurons using antibodies against the neuronal marker NeuN and Fos protein, a marker of strongly activated neurons. Until now, Fos-expressing neurons and other cell types were isolated from fresh tissue, which entailed long processing days and allowed very limited numbers of brain samples to be assessed after lengthy and complex behavioral procedures. Here we found that yields of Fos-expressing neurons and Fos mRNA from dorsal striatum were similar between freshly dissected tissue and tissue frozen at -80 &#186;C for 3 - 21 days. In addition, we confirmed the phenotype of the NeuN-positive and NeuN-negative sorted cells by assessing gene expression of neuronal (NeuN), astrocytic (GFAP), oligodendrocytic (Oligo2) and microgial (Iba1) markers, which indicates that frozen tissue can also be used for FACS isolation of glial cell types. Overall, it is possible to collect, dissect and freeze brain tissue for multiple FACS sessions. This maximizes the amount of data obtained from valuable animal subjects that have often undergone long and complex behavioral procedures.

Fluorescence Activated Cell Sorting FACS and Gene Expression Analysis of Fos-expressing Neurons from Fresh and Frozen Rat Brain Tissue

Here we present a Fluorescence Activated Cell Sorting (FACS) protocol to study molecular alterations in Fos-expressing neuronal ensembles from both fresh and frozen brain tissue. The use of frozen tissue allows FACS isolation of many brain areas over multiple sessions to maximize the use of valuable animal subjects.

The study of neuroplasticity and molecular alterations in learned behaviors is switching from the study of whole brain regions to the study of specific ...

Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a protein, it is vital to also determine its distribution. Here, we describe a protocol employing immunofluorescence, confocal microscopy, and computer-based analysis to determine the distribution of the synaptic protein Mover (also called TPRGL or SVAP30). We compare the distribution of Mover to that of the synaptic vesicle protein synaptophysin, thereby determining the distribution of Mover in a quantitative manner relative to the abundance of synaptic vesicles. Notably, this method could potentially be implemented to allow for comparison of the distribution of proteins using different antibodies or microscopes or across different studies. Our method circumvents the inherent variability of immunofluorescent stainings by yielding a ratio rather than absolute fluorescence levels. Additionally, the method we describe enables the researcher to analyze the distribution of a protein on different levels: from whole brain slices to brain regions to different subregions in one brain area, such as the different layers of the hippocampus or sensory cortices. Mover is a vertebrate-specific protein that is associated with synaptic vesicles. With this method, we show that Mover is heterogeneously distributed across brain areas, with high levels in the ventral pallidum, the septal nuclei, and the amygdala, and also within single brain areas, such as the different layers of the hippocampus.

Here, we describe a quantitative approach to determining the distribution of a synaptic protein relative to a marker protein using immunofluorescence staining, confocal microscopy, and computer-based analysis.

The presence, absence, or levels of specific synaptic proteins can severely influence synaptic transmission. In addition to elucidating the function of a ...

A Time-Efficient Fluorescence Spectroscopy-Based Assay for Evaluating Actin Polymerization Status in Rodent and Human Brain Tissues

Actin, the major component of cytoskeleton, plays a critical role in the maintenance of neuronal structure and function. Under physiological states, actin occurs in equilibrium in its two forms: monomeric globular (G-actin) and polymerized filamentous (F- actin). At the synaptic terminals, actin cytoskeleton forms the basis for critical pre- and post-synaptic functions. Moreover, dynamic changes in the actin polymerization status (interconversion between globular and filamentous forms of actin) are closely linked to plasticity-related alterations in synaptic structure and function. We report here a modified fluorescence-based methodology to assess polymerization status of actin in ex vivo conditions. The assay employs fluorescently labelled phalloidin, a phallotoxin that specifically binds to actin filaments (F-actin), providing a direct measure of polymerized filamentous actin. As a proof of principle, we provide evidence for the suitability of the assay both in rodent and post-mortem human brain tissue homogenates. Using latrunculin A (a drug that depolymerizes actin filaments), we confirm the utility of the assay in monitoring alterations in F-actin levels. Further, we extend the assay to biochemical fractions of isolated synaptic terminals wherein we confirm increased actin polymerization upon stimulation by depolarization with high extracellular K+.

We report a simple, time-efficient and high-throughput fluorescence spectroscopy-based assay for the quantification of actin filaments in ex vivo biological samples from brain tissues of rodents and human subjects.

Actin, the major component of cytoskeleton, plays a critical role in the maintenance of neuronal structure and function. Under physiological states, actin ...

Quantifying the Distribution of a Presynaptic Protein in the Mouse Brain Using Immunofluorescence

Source: Wallrafen, R., et al. <a href="https://app.jove.com/v/58940">Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence.</a> J. Vis. Exp. (2019).
This video demonstrates the immunofluorescence staining of a mouse brain slice to quantify the distribution of a specific presynaptic protein. The process involves blocking nonspecific sites, applying primary antibodies for the target protein and a reference marker, followed by secondary antibody labeling and nuclear counterstaining. The reference marker ensures accurate quantification by providing a consistent baseline, and the distribution of the presynaptic protein is analyzed across brain regions using confocal microscopy.

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

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JoVE Encyclopedia of Experiments

Neuropathology

Trauma

Measuring Protein Expression in the Rodent Brain Using Near-Infrared Fluorescence and High-Resolution Scanning 

Source:&#160;<a style='text-decoration:none;' href='https://app.jove.com/v/59685/using-near-infrared-fluorescence-and-high-resolution-scanning-to-measure-protein-expression-in-the-rodent-brain'><span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>Kimmelmann-Shultz, B.,&#160;et. al.<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>&#160;<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain.&#160;J. Vis. Exp.<span style='-webkit-text-decoration-skip:none;font-family:Arial,sans-serif;font-size:12pt;font-style:normal;font-variant:normal;font-weight:400;text-decoration-skip-ink:none;vertical-align:baseline;white-space:pre-wrap;'>&#160;(2019)</a> &#160;This video demonstrates a near-infrared fluorescence and high-resolution scanning method for measuring protein expression in the rodent brain.

Source:&#160;Kimmelmann-Shultz, B.,&#160;et. al.&#160;Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

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