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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 dynamics^3,4,5. 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 immunohistochemistry^6,7,8,9. 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 gephyrin^18,19,20 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 release^25,26,27. 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.

Protokół

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 Universitätsmedizin Göttingen) under the approval number T 10/30.

NOTE: For this protocol, 3 adult male C57BL/6 mice were used.

1. Sample Preparation

1. Prepare fixative and 0.1 M phosphate buffer (PB; see Table 1).
2. 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).
3. Open the skull with scissors and carefully isolate the brain using a spoon with blunt edges to avoid damaging the tissue.
4. Fill a 50 mL reaction tube with fixative and postfix the brain in 4% PFA at 4 °C overnight.
5. Remove the fixative and wash the brain in 50 mL of 0.1 M PB on a shaker for 30 min.
6. 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 °C for cryoprotection.
7. 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 °C freezer.
8. Mount the frozen tissue for sectioning. Equilibrate the tissue to the cryomicrotome temperature for at least 15 min before sectioning.
9. Section the brain into 25 µm 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 °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.

2. Immunofluorescence

1. Prepare solutions including the blocking buffer, antibody buffer, washing buffer 1, and washing buffer 2 (see Table 1).
2. Rinse slices once with PB to remove excess OCT.
   1. Remove the solution with a plastic pipette without sucking in the brain slices. Add 250 µL of fresh PB with a 1000 µL pipette.
      CAUTION: Slices should not dry out, so remove and add fluids well by well.
3. Remove the PB with a plastic pipette and add 250 µL of blocking buffer per well with a 1000 µL pipette. Incubate for 3 h at room temperature (RT) on the shaker.
4. During the incubation time, dilute the primary antibodies in antibody buffer in a reaction tube. Use 250 µL antibody buffer per well and add the appropriate amount of antibody (see Table 2) by pipetting it directly into the solution using a 2 µL 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.
5. After the incubation time, remove the blocking buffer with a plastic pipette and add 250 µL of antibody solution containing primary antibodies per well. Incubate slices with primary antibody overnight at 4 °C on a shaker.
6. Next day, wash the slices with washing buffer 1 3x for 10 min at RT on a shaker.
   1. Remove the medium with a plastic pipette and add 300 µL of washing buffer 1 per well. Incubate at RT for 10 min. Repeat 3 times.
7. During the washing steps, dilute the fluorophore-coupled secondary antibodies in antibody buffer in a reaction tube. Use 250 µL antibody buffer per well and add the appropriate amount of antibody (see Table 2) by pipetting it directly into the solution using a 2 µL 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.
8. After the washing steps, remove the washing buffer with a plastic pipette and add 250 µL of antibody solution containing secondary antibodies per well. Incubate the slices with secondary antibody for 90 min at RT in the dark.
9. Wash the slices with washing buffer 2 3x for 10 min at RT.
10. During the washing steps, dilute 4′,6-diamidino-2-phenylindole (DAPI) in 0.1 M PB in a concentration of 1:2000.
11. Remove the washing buffer 2 with a plastic pipette and add 250 µL of DAPI solution per well. Incubate for 5 min at RT on the shaker.
12. Remove the DAPI solution with a plastic pipette and add 500 µL of 0.1 M PB per well with a 1000 µL pipette.
13. Mount slices on microscope slides.
    1. 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.
    2. Use the fine brush to flatten and orient the slices on the microscope slide.
    3. When all slices are positioned correctly, remove excess PB with a tissue and dry the slide carefully.
       CAUTION: Avoid drying the brain slices completely.
    4. Add 80 µL of embedding medium onto the slide. Carefully lower the coverslip onto the slide, thereby embedding the brain slices.
    5. 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 °C.
       NOTE: The protocol can be paused here.

3. Imaging

1. 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.
2. 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.
3. Acquire virtual tissues of the whole brain slice for the different channels.
   1. In the imaging software (see Table of Materials), select the Tiles option and manually delineate the brain slice with the Tile Region Setup.
   2. Distribute support points throughout the tile region and adjust the focus for the different support points by pressing Verify Tile Regions/Positions….
   3. Adjust the settings in Acquisition Mode according to the desired resolution and file size of the resulting image and start the scan.
4. 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.

4. Computer-based Analysis

1. Load all single channels for one image into FIJI²⁹ by clicking File| Open.
2. With the Freehand selection tool, delineate one hemisphere in the DAPI-channel. Create a mask of the selection by clicking Edit| Selection| Create mask.
3. 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.
4. Copy the mean fluorescence intensity for the single channels into a spreadsheet.
5. 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.
6. 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).

5. Data Handling

1. 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.
2. 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.
3. 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.
4. 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.

Wyniki

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...

Dyskusje

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...

Materiały

Name	Company	Catalog Number	Comments
1.5 mL reaction tubes	Eppendorf	30120094
50 mL reaction tubes	Greiner Bio-One	227261
multiwell 24 well	Fisher Scientific	087721H
plastic pipette (disposable)	Sarstedt	861,176
1000 mL pipette	Rainin	17014382
2 ml pipette	Eppendorf	3123000012
Vortex Genius 3	IKA	3340001
Menzel microscope slides	Fisher Scientific	10144633CF
Stereoscope	Leica
LSM800	Zeiss		Confocal microscope
freezing microtome	Leica
PBS (10X)	Roche	11666789001
PFA	Sigma	P6148-1kg
NaCl	BioFroxx	1394KG001
sucrose	neoFroxx	1104KG001
Tissue Tek	Sakura	4583	OCT
Na2HPO4	BioFroxx	5155KG001
NaH2PO4	Merck	1,063,460,500
normal goat serum	Merck Millipore	S26-100ML
normal donkey serum	Merck	S30-100ML
Triton X-100	Merck	1,086,031,000
rabbit anti-Mover	Synaptic Systems	RRID: AB_10804285
guinea pig anti-Synaptophysin	Synaptic Systems	RRID: AB_1210382
donkey anti-rabbit AF647	Jackson ImmunoResearch	RRID: AB_2492288
goat anti-mouse AF488	Jackson ImmunoResearch	RRID: AB_2337438
Mowiol4-88	Calbiochem	475904
ZEN2 blue software	Zeiss		Microscopy software
FIJI	ImageJ		Analysis software
Microsoft Excel	Microsoft