Name: Quantifying the Heterogeneous Distribution of a Synaptic Protein in the Mouse Brain Using Immunofluorescence (Video) | JoVE Journal
Uploaded: 2019-01-29T13:00:00.000+00:00
Duration: 9 min 18 s
Description: Learn to quantify synaptic protein distribution in mouse brain using immunofluorescence and confocal microscopy. This ratio-based method analyzes heterogeneity across brain regions and adapts to various tissues and model systems.

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5 minutes

We thank Irmgard Weiss for excellent technical assistance. The authors acknowledge support by Hermes Pofantis and Andoniya Petkova. The authors also thank the European Neuroscience Institute for the usage of the LSM800 and technical assistance, especially by Dr. Nils Halbsgut. This work was funded by the University Medical Center G&#246;ttingen. JSV acknowledges support by the Center for Nanoscale Microscopy and Molecular Physiology of the Brain (CNMPB).

This protocol does not involve experiments on live animals. Experiments involving euthanizing of animals to obtain brain samples were approved by the local animal protection authorities (Tierschutzkommission der Universit&#228;tsmedizin G&#246;ttingen) under the approval number T 10/30.
NOTE: For this protocol, 3 adult male C57BL/6 mice were used.
1. Sample Preparation
<ol>
	<li>Prepare fixative and 0.1 M phosphate buffer (PB; see Table 1).</li>
	<li>Fix the animal by transcardial perfusion as described in Gage et al.28. First wash out the blood with 0.9% NaCl-solution, then perfuse with 30 mL of 4% paraformaldehyde (PFA).</li>
	<li>Open the skull with scissors and carefully isolate the brain using a spoon with blunt edges to avoid damaging the tissue.</li>
	<li>Fill a 50 mL reaction tube with fixative and postfix the brain in 4% PFA at 4 &#176;C overnight.</li>
	<li>Remove the fixative and wash the brain in 50 mL of 0.1 M PB on a shaker for 30 min.</li>
	<li>After washing, incubate the brain in a 50 mL reaction tube in 30% sucrose in 0.1 M PB for 48 h or until it sinks in the tube at 4 &#176;C for cryoprotection.</li>
	<li>Trim the cryoprotected brain with a sharp blade, place it in a cryomold, and embed it with optimal cutting temperature (OCT) compound. Avoid bubbles. Orient the brain and freeze the cryomold in the -80 &#176;C freezer.</li>
	<li>Mount the frozen tissue for sectioning. Equilibrate the tissue to the cryomicrotome temperature for at least 15 min before sectioning.</li>
	<li>Section the brain into 25 &#181;m thick coronal slices. Touch the OCT carefully with a glass hook without touching the brain tissue. Collect 3 adjacent slices per well in a 24 well plate and store them in 0.1 M PB at 4 &#176;C until staining. 
	NOTE: The protocol can be paused here for up to two weeks. Longer storage times can interfere with the tissue quality and thus influence the outcome of the experiment.</li>
</ol>
2. Immunofluorescence
<ol>
	<li>Prepare solutions including the blocking buffer, antibody buffer, washing buffer 1, and washing buffer 2 (see Table 1).</li>
	<li>Rinse slices once with PB to remove excess OCT.
	<ol>
		<li>Remove the solution with a plastic pipette without sucking in the brain slices. Add 250 &#181;L of fresh PB with a 1000 &#181;L pipette. 
		CAUTION: Slices should not dry out, so remove and add fluids well by well.</li>
	</ol>
	</li>
	<li>Remove the PB with a plastic pipette and add 250 &#181;L of blocking buffer per well with a 1000 &#181;L pipette. Incubate for 3 h at room temperature (RT) on the shaker.</li>
	<li>During the incubation time, dilute the primary antibodies in antibody buffer in a reaction tube. Use 250 &#181;L antibody buffer per well and add the appropriate amount of antibody (see Table 2) by pipetting it directly into the solution using a 2 &#181;L pipette. Mix the solution by gently pipetting up and down several times. Vortex shortly afterwards to ensure proper mixing. 
	NOTE: To determine the background fluorescence, stainings should also be performed without adding the primary antibody. For that, incubate the slice in antibody solution without primary antibodies according to the protocol.</li>
	<li>After the incubation time, remove the blocking buffer with a plastic pipette and add 250 &#181;L of antibody solution containing primary antibodies per well. Incubate slices with primary antibody overnight at 4 &#176;C on a shaker.</li>
	<li>Next day, wash the slices with washing buffer 1 3x for 10 min at RT on a shaker.
	<ol>
		<li>Remove the medium with a plastic pipette and add 300 &#181;L of washing buffer 1 per well. Incubate at RT for 10 min. Repeat 3 times.</li>
	</ol>
	</li>
	<li>During the washing steps, dilute the fluorophore-coupled secondary antibodies in antibody buffer in a reaction tube. Use 250 &#181;L antibody buffer per well and add the appropriate amount of antibody (see Table 2) by pipetting it directly into the solution using a 2 &#181;L pipette. Mix the solution by gently pipetting up and down several times. Vortex shortly afterwards to ensure proper mixing. 
	CAUTION: Because the antibodies are light-sensitive, all steps from this point on need to be performed in the dark.</li>
	<li>After the washing steps, remove the washing buffer with a plastic pipette and add 250 &#181;L of antibody solution containing secondary antibodies per well. Incubate the slices with secondary antibody for 90 min at RT in the dark.</li>
	<li>Wash the slices with washing buffer 2 3x for 10 min at RT.</li>
	<li>During the washing steps, dilute 4&#8242;,6-diamidino-2-phenylindole (DAPI) in 0.1 M PB in a concentration of 1:2000.</li>
	<li>Remove the washing buffer 2 with a plastic pipette and add 250 &#181;L of DAPI solution per well. Incubate for 5 min at RT on the shaker.</li>
	<li>Remove the DAPI solution with a plastic pipette and add 500 &#181;L of 0.1 M PB per well with a 1000 &#181;L pipette.</li>
	<li>Mount slices on microscope slides.
	<ol>
		<li>Place a microscope slide under the stereoscope. With a fine brush, add three separate drops of 0.1 M PB onto the slide. Place one slice per drop onto the microscope slide.</li>
		<li>Use the fine brush to flatten and orient the slices on the microscope slide.</li>
		<li>When all slices are positioned correctly, remove excess PB with a tissue and dry the slide carefully. 
		CAUTION: Avoid drying the brain slices completely.</li>
		<li>Add 80 &#181;L of embedding medium onto the slide. Carefully lower the coverslip onto the slide, thereby embedding the brain slices.</li>
		<li>Leave the slides to dry in the fume hood for 1-2 h (cover them to avoid light exposure) and store them in a microscope slide box at 4 &#176;C. 
		NOTE: The protocol can be paused here.</li>
	</ol>
	</li>
</ol>
3. Imaging
<ol>
	<li>After the embedding medium is completely hardened, place the microscope slide under the confocal microscope. 
	NOTE: Epifluorescence microscopy combined with deconvolution software should yield similar image quality.</li>
	<li>Adjust the laser settings by increasing or decreasing the laser intensity for every channel so that few pixels are overexposed to ensure maximum distribution of grey values.</li>
	<li>Acquire virtual tissues of the whole brain slice for the different channels.
	<ol>
		<li>In the imaging software (see Table of Materials), select the Tiles option and manually delineate the brain slice with the Tile Region Setup.</li>
		<li>Distribute support points throughout the tile region and adjust the focus for the different support points by pressing Verify Tile Regions/Positions&#8230;.</li>
		<li>Adjust the settings in Acquisition Mode according to the desired resolution and file size of the resulting image and start the scan.</li>
	</ol>
	</li>
	<li>When the scan is finished, use the Stitching function to process the virtual tissue. Export the file as a .tif with the function Image Export.</li>
</ol>
4. Computer-based Analysis
<ol>
	<li>Load all single channels for one image into FIJI29 by clicking File| Open.</li>
	<li>With the Freehand selection tool, delineate one hemisphere in the DAPI-channel. Create a mask of the selection by clicking Edit| Selection| Create mask.</li>
	<li>Determine the mean fluorescence intensity for the single channels (Mover and synaptophysin) by clicking Analyze| Measure. 
	NOTE: Make sure to select the different channels to determine the mean fluorescence intensity values for each channel.</li>
	<li>Copy the mean fluorescence intensity for the single channels into a spreadsheet.</li>
	<li>Determine the mean fluorescence intensity for the single channels in an area of interest by delineating the area also with the Freehand selection tool. Use a mouse brain atlas as reference.</li>
	<li>Repeat steps 4.1-4.5 for all hemispheres and all areas of interest. 
	NOTE: Determine the values for each hemisphere separately in order to later compare the values in an area of interest to that in the hemisphere (see step 5.2).</li>
</ol>
5. Data Handling
<ol>
	<li>In case the background fluorescence is high (see Discussion), a background subtraction might be needed. For that, determine the mean fluorescence intensity for the slice processed without primary antibody against the reference protein (here: synaptophysin) and subtract that value from all values obtained for the brain regions and hemispheres.</li>
	<li>When the mean fluorescence intensities for the single channels for every hemisphere and every area of interest have been determined (see Table 3), calculate the ratio of Mover to synaptophysin by dividing the value for Mover by the value for synaptophysin (yellow in Table 3). Perform this action for every hemisphere and every area of interest separately.</li>
	<li>Divide the ratio obtained for one area of interest by the ratio obtained for the corresponding hemisphere (orange in Table 3) to determine the ratio of the area of interest to the hemisphere.</li>
	<li>To determine the relative Mover abundance, translate the ratio obtained in 5.2 into a percentage by determining its deviation from 1 (red in Table 3). A ratio of 1.25 would therefore give a relative Mover abundance of 25% above average, and a ratio of 0.75 would yield a relative Mover abundance of 25% below average.</li>
</ol>

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</ol>

The authors have nothing to disclose.

The method presented here aims at quantifying the distribution of a protein of interest relative to the abundance of a marker protein with a known distribution. Immunofluorescence staining can show a high variability of staining intensities between different slices. The quantification approach described here circumvents this problem by determining the ratio of the protein of interest to the average across the hemisphere. Therefore, different staining intensities across slices are cancelled out and allow for a quantitativ...

Communication between neurons happens at specialized contact sites called synapses. Synapses contain a myriad of different proteins that orchestrate synaptic transmission. Some of those proteins show a heterogeneous distribution throughout the nervous system and are not present in every synapse1. One example for such a protein is Munc13, which is involved in the priming process of synaptic vesicles. There are different isoforms of Munc13, which are heterogeneously distributed throughout the brain2, and the presence or absence of specific isoforms can influence short-term synaptic plasticity and synaptic vesicle dynamics3,4,5. Therefore, it is of vital importance to be able to identify the presence of different synaptic proteins across brain areas.
The methods of choice for quantification of synaptic proteins - so far - are mass spectrometry and Western blotting, rather than immunohistochemistry6,7,8,9. In some cases, several methods are used to complement each other to assess both the quantity and the localization of specific proteins (i.e., Wilhelm et al.10). The method we describe here allows for the localization and quantification of proteins of interest without the need of using any biochemical method, simply employing immunofluorescent stainings. Another advantage here is that the quantification can be done over areas much smaller and, therefore, more specific, than those achieved by other methods. However, one has to take into consideration that a reliable reference protein is needed to assess the distribution of the protein of interest.
Fluorescent staining by immunohistochemistry allows us to routinely identify the localization of proteins across brain areas as well as within different neuronal compartments. To identify the different compartments, specific markers are used. Typically, antibodies against synapsin and synaptophysin11 can be used to label synaptic vesicles, while antibodies against bassoon label the active zone of a presynaptic terminal12. Vesicular transporters, such as the vesicular glutamate transporters (vGluT) or vesicular GABA transporter (vGAT), are used to label excitatory13 and inhibitory14 presynaptic terminals, respectively. On the postsynaptic side, antibodies against the Homer protein can be employed to mark postsynaptic terminals, and antibodies against postsynaptic density protein 95 (PSD95)15,16,17 or gephyrin18,19,20 can label excitatory or inhibitory postsynaptic terminals, respectively. By using antibodies against a protein of interest and markers such as the ones described above, one can determine the localization of such protein. Many studies to date have done this in a qualitative manner21. However, to reliably determine the differential distribution of a specific synaptic protein, one must not only determine its presence or absence but also its relative concentration. The heterogeneity of sizes and density of synapses makes it important to establish a ratio between the synaptic marker and the protein of interest. Otherwise, synapse-rich regions such as the non-pyramidal layers of the hippocampus and the molecular layer of the cerebellum will show a high density of synaptic proteins, only due to the higher density of synapses but not due to a strong presence of that protein in each synapse (e.g., Wallrafen and Dresbach1). On the other hand, proteins in the neuronal soma (e.g., TGN3822) will usually show strong presence in the hippocampal pyramidal cell layer or hippocampal or cerebellar granule cell layer due to the high concentration of neuronal cell bodies in those areas. Therefore, this non-homogeneous distribution of structures, in this case synapses, can lead to a false estimation of the distribution of the protein of interest itself. Furthermore, there is an intrinsic variability in staining intensities across samples in immunohistochemical stainings. The protocol described here takes this into consideration and avoids such biases, as well as other caveats that arise from immunohistochemical methods.
In our recent study, we have used this method to describe the differential expression of Mover (also called TPRGL23 or SVAP3024) across 16 different brain areas1. Mover is a vertebrate-specific synaptic protein that can be found in association to synaptic vesicles and influences neurotransmitter release25,26,27. We have related the Mover expression to the abundance of synaptic vesicles, by staining for synaptophysin as a synaptic vesicle reference marker. We found high levels of Mover particularly in the septal nuclei, the ventral pallidum, and the amygdala. Within the hippocampus, we found a heterogeneous distribution of Mover, with high levels in the layers associated with intra-hippocampal computation, and low levels in input- and output layers.

<table><tbody><tr><td>1.5 mL reaction tubes</td><td>Eppendorf</td><td>30120094</td><td></td></tr><tr><td>50 mL reaction tubes</td><td>Greiner Bio-One</td><td>227261</td><td></td></tr><tr><td>multiwell 24 well</td><td>Fisher Scientific</td><td>&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 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475904</td><td></td></tr><tr><td>ZEN2 blue software</td><td>Zeiss</td><td></td><td>Microscopy software</td></tr><tr><td>FIJI</td><td>ImageJ</td><td></td><td>Analysis software</td></tr><tr><td>Microsoft Excel</td><td>Microsoft</td><td></td><td></td></tr></tbody></table>

quantifying the heterogeneous distribution of a synaptic protein in the mouse brain using immunofluorescence

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.

Representative staining patterns of different markers can be seen in Figure 1. The pattern varies depending on the distribution of the protein. Examples of five rostro-caudal levels are shown in columns (A)-(E). A representative DAPI staining is shown in the first row: DAPI adheres to the DNA of a cell and thus nuclei are stained. This results in a punctate pattern. Regions with a high cell density are brighter than regions w...

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 ...

quantifying-heterogeneous-distribution-synaptic-protein-mouse-brain

Research

JoVE Journal

Neuroscience

This protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers.

Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

The abundance of neurotransmitter receptors clustered at synapses strongly influences synaptic strength. This method quantifies fluorescently-labeled neurotransmitter receptors in three dimensions with single-synapse resolution in C. elegans, allowing hundreds of synapses to be rapidly characterized within a single sample without distortions introduced by z-plane projection.

Automated Quantification of Synaptic Fluorescence in C. elegans

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...

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

Source: Schönherr, S., et. al. Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala. J. Vis. Exp. (2016)This video demonstrates the immunolabelling of freeze-fractured replicas of mouse brain tissue for electron microscopy. The fractured and coated replicas are digested to expose receptors. Primary and gold-tagged secondary antibodies are added to label specific neuronal membrane receptors. The replica...

Immunolabeling of Neuronal Membrane Proteins in a Freeze-fractured Specimen of Mouse Brain Tissue

Source: McLeod, F., et al. Evaluation of Synapse Density in Hippocampal Rodent Brain Slices. J. Vis. Exp. (2017).This video demonstrates a method for visualizing synaptic markers in hippocampal brain slices using confocal immunofluorescence analysis. The slices are immunostained with primary antibodies targeting presynaptic and postsynaptic markers, followed by fluorophore-conjugated secondary antibodies. The labeled markers are then visualized using confocal microscopy to evaluate the synaptic...

Microscopic Analysis of Synapses in Mouse Hippocampal Slices Using Immunofluorescence

This article illustrates how the expression of neurotransmitter receptors can be quantified and the pattern analyzed at synapses with identified pre and postsynaptic elements using a combination of viral transduction of optogenetic tools and the freeze-fracture replica immunolabeling technique.

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

We describe an optical assay for synaptic vesicle (SV) recycling in cultured neurons. Combining this protocol with double transfection to express a presynaptic marker and protein of interest allows us to locate presynaptic sites, their synaptic vesicle recycling capacity, and determine the role of the protein of interest.

An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins

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

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

This protocol describes a method for combining fluorescence in situ hybridization (FISH) and fluorescence immunohistochemistry (IHC) in both fresh frozen and fixed mouse brain sections, with the goal of achieving multilabel FISH and fluorescence IHC signal. IHC targeted cytoplasmic and membrane attached proteins.

Combining Multiplex Fluorescence In Situ Hybridization with Fluorescent Immunohistochemistry on Fresh Frozen or Fixed Mouse Brain Sections

This protocol presents a robust, detailed method to obtain highly pure synaptosomes, synaptic vesicles, and other synaptic fractions from the mouse brain. This method enables the evaluation of synaptic processes, including the biochemical analysis of protein localization and function with compartmental resolution.

Subcellular Fractionation for the Isolation of Synaptic Components from the Murine Brain

Learn to quantify synaptic protein distribution in mouse brain using immunofluorescence and confocal microscopy. This ratio-based method analyzes heterogeneity across brain regions and adapts to various tissues and model systems.

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

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