This work was supported by the MRC, EU FP7, ARUK, Wellcome Trust, and Parkinson&#39;s UK. We would also like to thank Ms. Johanna Buchler for her contribution of confocal images and the members of the lab for their constructive input on the manuscript and methodology.

All the experiments using mice and rats were approved and conducted using Schedule 1 procedures covered under the Home Office Animals (Scientific Procedures) act 1986. The artificial cerebrospinal fluid (ACSF) recipe can be found in Table 1.
1. Acute Hippocampal Slice Preparation
<ol>
	<li>Remove the mouse brain as quickly as possible, using a spatula and sharp scissors to cut the skull, and place it in ice-cold, oxygenated (95% O2, 5% CO2), high-sucrose ACSF (~100 mL).</li>
	<li>Place the mouse brain on a Petri dish and remove the cerebellum and a small section of the frontal cortex with a scalpel before hemisecting down the midline of the brain.</li>
	<li>Place the two hemispheres on the side that was just cut and glue each hemisphere onto the stage of a vibratome.</li>
	<li>Cut 200 - 300 &#181;m-thick sections of the complete region of interest. In the case of the hippocampus, approximately 3 - 4 slices per hemisphere should be obtained.</li>
	<li>Using a plastic Pasteur pipette, transfer the slices to a chamber submerged in oxygenated (95% O2, 5% CO2) ACSF. Maintain at 34 &#176;C for 30 min. 
	NOTE: The treatment of slices with active pharmacological agents, such as recombinant Dkk1 proteins, can be performed at this stage by adding the agents to the slice chamber.</li>
	<li>Remove the slices from the chamber using a paintbrush, place them into a 24-well plate, and fix for 20 min to 1 h in 4% paraformaldehyde (PFA)/4% sucrose at room temperature (RT). 
	Caution: PFA is toxic, wear appropriate protection.</li>
	<li>Wash the slices three times in 1X PBS (10 min each). 
	NOTE: The protocol can be paused here for 1 - 2 days, as long as the slices are kept at 4 &#176;C in 1X PBS.</li>
</ol>
2. Immunofluorescence for Synaptic Markers
NOTE: The recipes of all buffers used can be found in Table 2.
<ol>
	<li>Replace the PBS with blocking/permeabilizing buffer (Table 2) in the slice wells and incubate at RT for 4 - 6 h.</li>
	<li>Dilute the polyclonal guinea pig antibody against vGlut1 at a 1:2,000 dilution in blocking/permeabilization buffer. 
	NOTE: vGlut1 is a marker for pre-synaptic excitatory terminal visualization. For post-synaptic excitatory synapse identification, use a polyclonal antibody against PSD-95 and dilute 1:500 in the same solution. Use a minimum volume of 300 &#181;L in each well. To identify the anatomical location of the hippocampus, use an antibody that can also identify the neuronal structure, such as MAP2 or Tubulin. Work out the correct concentrations of all primary antibodies for the synaptic markers of choice (pre- and post-synaptic) and dilute in fresh blocking/permeabilizing buffer.</li>
	<li>Incubate the slices in the primary antibody solution overnight (or for 1 - 2 days) at 4 &#176;C. Use a shaking platform with vigorous movement.</li>
	<li>Wash the slices three times in PBS (10 min each).</li>
	<li>Dilute the appropriate secondary antibodies 1:500 in blocking buffer. For example, when using the vGlut1 guinea pig antibody, apply a secondary antibody that recognizes the guinea pig component (e.g., donkey anti-guinea-pig) conjugated to a specific fluorophore. When using the PSD-95 rabbit antibody, apply a secondary antibody that recognizes the rabbit component (e.g., donkey anti-rabbit) conjugated to a different specific fluorophore.</li>
	<li>Incubate the slices in this solution for 2 - 3 h at RT. Ensure that the slices are protected from the light, as secondary antibodies are light sensitive.</li>
	<li>Wash the slices three times in PBS (10 min each).</li>
	<li>Carefully remove the slices from the 24-well plate using a paintbrush and place them evenly onto pre-labelled glass slides. Add a drop of mounting medium on top of each slice and then gently place a glass coverslip on top of the slices. Avoid the formation of air bubbles. Take care to use enough mounting medium (300-400 &#181;L), as an insufficient amount may lead to dry slices.</li>
	<li>Leave to dry for a minimum of 1 - 2 days at RT and keep the slides protected from light.</li>
	<li>Store the slides at 4 &#176;C in the short-term, but for long-term storage, keep them at -20 &#176;C.</li>
</ol>
3. Confocal Image Acquisition and Analysis
<ol>
	<li>Imaging using confocal laser scanning microscopy.
	<ol>
		<li>Identify the region of the hippocampus to be imaged (e.g., the cornu ammonis 1 and 3 (CA1, CA3) or the dentate gyrus (DG)) by means of a 10x or 20x objective.</li>
		<li>Change to a 40x or 63x oil-immersion objective to make sure the slice anatomy is intact by identifying continuous neurites and organized structure. Use a neuronal marker, such as MAP2, as a reference (Figure 1A).</li>
		<li>Subsequently, switch to a 60x oil-immersion objective (NA = ~1.3 - 1.4) and adjust the settings for each channel to obtain optimal signal and contrast. Set the intensity of each laser to avoid the saturation of any pixels using a high-/low-contrast option. 
		NOTE: These setting will depend on the microscope used, but refer to the Table of Materials for the laser power settings used in Figure 2, Figure 3, and Figure 4. For best results, use a 1024 x 1024-pixel resolution. The settings should remain constant throughout the different conditions of the same experiment. Additional zoom can be applied if required.</li>
		<li>Evaluate the depth where the staining is even and acquire image stacks of at least 8 equidistant (250 nm) planes. Then take 3 adjacent representative images stacks from the same area of interest per slice. 
		NOTE: The depth may vary from one slice to another but should always be approximately 2 - 5 &#181;m from the surface of the slice.</li>
		<li>Repeat the acquisition in at least 3 slices per condition and from 6 - 8 animals per treatment group.</li>
	</ol>
	</li>
	<li>Image analysis. 
	NOTE: Use an automated image analysis software (See the Table of Materials). Note that some systems require a license, whereas others are free, such as ImageJ. The software used must analyze the images in 3D by taking into account all planes of the stack imaged. Refer to the Table of Materials for the details of the software used in the image analysis (Figure 1).
	<ol>
		<li>Use a custom-based intensity threshold protocol to identify all synaptic puncta and neuronal markers, which are manually optimized and selected within the software [see Table of Materials for the specific software used in this protocol]. 
		NOTE: The software identifies a minimum and maximum intensity for each fluorophore and associates a percentage of intensity for each recognized object. Note that the percentage of intensity will vary according to the fluorophore used and the antibody against the synaptic marker.</li>
		<li>For synaptic markers, ensure that size filters (&#62;0.1&#181;m3 and &#60;0.8&#181;m3) are selected within the software and applied to the image. Adjust the parameters to ensure that the selected objects are not overlapping. Exclude any object that are too large or too small to be considered synaptic puncta (see Figure 2B). 
		NOTE: Once optimized, the same threshold values are then applied to all images within a given experiment.</li>
		<li>Quantify the mean number, intensity, and volume of individual synaptic puncta (pre- or post-synaptic) using the optimized threshold protocols selected within the software. Normalize the puncta number to the volume of the image field (roughly 16,928 &#181;m3 in the system used here). Normalize the synaptic puncta intensity to the intensity of the reference neuronal marker. 
		NOTE: Synapses are defined as the co-localization between both pre- and post-synaptic markers. Once the threshold protocols for pre- and post-synaptic puncta have been established, the software should identify the co-localization of synaptic puncta as the overlap of 1 pixel or more in a single plane. 
		NOTE: For statistical analysis, confirm that all sets of samples follow normality and homogeneity of variance, as determined by Lilliefords and &#967;2-tests, respectively. Samples showing a normal distribution and homogeneity of variance should be analyzed with parametric tests, as described below. For example, excitatory synaptic puncta analysis should be performed using a one-way ANOVA with blocking and replication. Each individual experiment should be considered as a block; typically, there are three independent experiments, each with 1 - 3 mice from each condition.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56153/56153fig1.jpg" /> 
Figure 1: Analysis of synaptic puncta using the image analysis software.&#160;(A) Screenshot of the intensity threshold protocol customization. The example given is for PSD-95, in which the selected puncta have a defined size of &#62;0.1 &#181;m3 and &#60;0.8 &#181;m3 and present minimized overlapping objects. Note that the image shown is for one confocal plane to enable the easier visualization of synaptic puncta and accurate parameter selection. (B) Screenshot of the analysis output from the software (following the selection of the optimized protocol). An example of raw synaptic puncta number measurements based on the volume. These values are then exported to a spreadsheet software. <a href="//cloudfront.jove.com/files/ftp_upload/56153/56153fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56153/56153fig2.jpg" /> 
Figure 2: Quality check of the slice and parameter settings for synaptic puncta. (A) Representative confocal images (40x objective) of the region of interest: the CA1 stratum radiatum (sr) and stratum oriens (so) of a hippocampal slice are identified by MAP2 staining. Scale bar =&#160;2 &#181;m. (B) Confocal images (60x objective and an additional 1.3X magnification on the acquisition software) of the CA1 stratum radiatum, with excitatory markers-vGlut1 (green) and PSD-95 (red)-from hippocampal slices. Note the identification of clear pre- and post-synaptic puncta. Puncta larger than 0.8 &#181;m3 are excluded from the quantification (white arrows). Scale bar =&#160;2 &#181;m (C) Confocal images (60x objective and an additional 1.3X magnification on the acquisition software) of the CA1 stratum radiatum, with excitatory markers-vGlut1 (green)-from cryostat-cut hippocampal sections from entire brains fixed in PFA. Note the presence of large holes and irregular, clumped vGlut1 staining. Scale bar =&#160;2 &#181;m. <a href="//cloudfront.jove.com/files/ftp_upload/56153/56153fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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The authors have nothing to disclose.

The precise evaluation of synapse number is essential to the field of neuroscience. Here, we show how to evaluate the density of synapses in the CA1 stratum radiatum region of hippocampal slices as a model system. The significance with respect to existing methods is demonstrated in our recent research article on the effect of Wnt deficiency in the hippocampus and where we also evaluated synapse number by single-section EM10. The magnitude of synapse loss are similar between single-section EM and the brain-slice method described here10. Thus, this finding demonstrates the accuracy and reliability of the technique.
Importantly, a limitation of both single-section EM and immunostaining synapses, as presented here, is the inability to accurately estimate the absolute synapse number for a specific brain region. Indeed, it is important to identify any global morphological changes, as changes in total volume could impact synaptic number. Another limitation is that certain antibodies are less efficient at staining intact brain regions, partially due to the extreme density of synaptic connections. We experienced higher-quality staining of vGlut1 and gephyrin using this slice preparation and subsequent PFA fixation for a maximum of 1 h, compared to immediate fixation of the entire brain in PFA (Figure 2C). The latter led to clumped synaptic staining, with holes in the tissue. Nonetheless, the vGlut1 antibody penetration is still restricted in both methods (a couple of tens of microns). We did not overcome this limitation, even with increased antibody incubation, but providing the antibody penetration is greater than the total depth that is imaged, there should be no influence on the final result. Furthermore, special care should be taken to ensure that the staining is even throughout the entire acquired stack.
In our study, we wanted to distinguish excitatory from inhibitory synapses. Consequently, we chose vGlut1/PSD-95 and vGAT/gephyrin, respectively, as these proteins are highly expressed in the adult mouse hippocampus and are specific to each synapse subtype4,5. Other excitatory and inhibitory synaptic markers could have been used to identify each respective synapse, such as receptors enriched at each synapse, including NMDA and AMPA receptors for excitatory and GABAA receptors for inhibitory synapses. Other neurotransmitters or neuromodulators can also be identified with our protocol, ensuring that specific antibodies are available, as shown for dopaminergic synapses in the striatum22. In addition, reducing the thickness of each slice to 120 &#181;m can partially overcome the low quantity of samples obtained with acute slices, which is imperative for studies in small brain structures such as the amygdala.
Future applications of our protocol could be implemented in the study of different brain areas and disorders. For example, in the striatum (a region of the brain associated with Parkinson&#39;s disease), a combination of vGlut2/PSD-95 or vGlut1/PSD-95 could be used to differentiate between the excitatory synapses from afferents coming from the cortex or thalamus, respectively22.
In conclusion, we have shown a reliable method to examine the number of synapses in brain tissues using pre- and post-synaptic markers to define a synapse. Critical steps include the preparation of good hippocampal slices, a fixation time of up to 1 h in PFA, the application of enough mounting medium, the use of reliable antibodies, appropriate image acquisition settings, and stringent synaptic puncta detection. Ultimately, this method is relatively quick and is easily reproducible by any laboratories that have access to a confocal microscope.

The human brain is composed of approximately 1014 synapses. Synapse number is tightly regulated during the development and maturation of the central nervous system (CNS). Throughout development, synapse number is modulated by synaptogenesis and pruning to form functional neuronal networks. Learning and memory are supported by the regulation of synapse number and strength, a process known as synaptic potentiation and depression1. Importantly, the stabilization of mature synapses is essential for maintaining neuronal network connections. Furthermore, deficits in synapse density have been reported at the early stages of neurodegenerative conditions such as Alzheimer&#39;s disease (AD)2. Therefore, the ability to accurately identify and evaluate synapse number is fundamental to the assessment of brain physiology and pathology.
The extreme density and compact nature of synapses make it challenging to estimate their precise number in intact brain areas. Chemical synapses in the CNS are made of two neurons in close apposition, separated by a synaptic cleft3. The pre-synaptic terminal, or synaptic bouton, emerges from an axon and consists of accumulated vesicles, which contain neurotransmitters that define the specificity of a synapse&#8212;namely, glutamate and gamma-aminobutyric acid (GABA) for excitatory and inhibitory synapses, respectively. The membranes of the vesicles contain vesicular transporters specific to each neurotransmitter: vesicular glutamate transporter (vGlut) for glutamate and vesicular GABA transporter (vGAT) for GABA. The post-synaptic terminal is a highly dense structure composed of multiple proteins, including receptors, adhesion molecules, and scaffold proteins. In excitatory synapses, post-synaptic density-95 protein (PSD-95) is the most abundant scaffold protein4, modulating excitatory N-methyl-D-aspartate(NMDA-) and &#945;-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA-) receptor function, whereas gephyrin anchors inhibitory receptors to the inhibitory post-synaptic terminal5. Overall, the specificity of proteins to pre- and post-synaptic compartments aid in the differentiation and identification of synapse subtypes.
The evaluation of synapse number can be performed with different techniques. The most common is the use of electron microscopy (EM), which enables the analysis of the synapse in compact and intact brain areas with high magnification and resolution. However, this technique is very expensive, requires complicated sample preparation, and is very time consuming. Other techniques include the use of array tomography6, which has a major disadvantage in that it requires specialized and expensive equipment to perform the analysis. Furthermore, transgenic lines expressing specific synaptic markers are useful at evaluating synapse number7, but the generation of new lines can be restrictive and costly, and the overexpression of proteins may result in undesirable off-target phenotypes.
Due to these limitations and for simplicity, many researchers evaluate synapse number by examining total synaptic protein levels. However, synapse loss can be observed before substantial changes in protein, as structural synaptic remodeling results in the dispersal of pre- or post-synaptic apposition, with no degradation of synaptic proteins. Furthermore, in AD, synaptic protein levels are reduced following synapse degeneration or neuronal death8,9. The quantification of total levels does not enable this distinction. Thus, there is a need to develop a reliable, accessible, and accurate method for the evaluation of synapse number in the brain.
In this article, we describe how to thoroughly identify and determine the number of synapses using immunofluorescence microscopy in hippocampal rodent brain slices. The synapses are defined by the colocalization of pre- and post-synaptic markers which appear in a punctate distribution.&#160;We demonstrate the validity of this technique through the study of Wnt signaling in the brain. Wnts are secreted proteins important for the formation, elimination, and maintenance of synapses. The in vivo induction of a secreted antagonist of the Wnt cascade, Dickkopf-1 (Dkk1), leads to excitatory synaptic loss while preserving inhibitory synapses in the hippocampus10. We illustrate the identification and count of excitatory and inhibitory synapses in hippocampal slices from an adult mouse expressing Dkk1 and highlight the transferability of this technique to other types of tissue/culture preparations.

<table><tbody><tr><td>Vibratome</td><td>Leica</td><td>VT1000S</td><td></td></tr><tr><td>Primary antibody</td><td>Millipore</td><td>AB5905</td><td>vGlut1 (1:2000)</td></tr><tr><td>Primary antibody</td><td>Thermo Scientific</td><td>MA1-046</td><td>PSD-95 (1:500)</td></tr><tr><td>Primary antibody</td><td>Synaptic Systems</td><td>131 004</td><td>vGAT (1:500)</td></tr><tr><td>Primary antibody</td><td>Synaptic Systems</td><td>147011</td><td>Gephyrin (1:500)</td></tr><tr><td>Primary antibody</td><td>Abcam</td><td>ab5392</td><td>MAP2 (1:10000)</td></tr><tr><td>Secondary antibody</td><td>Jackson Laboratories</td><td>706-545-148</td><td>Donkey Anti-Guinea Pig Alexa Fluor 488 (1:600)</td></tr><tr><td>Secondary antibody</td><td>Abcam</td><td>ab175470</td><td>Donkey Anti-Rabbit Alexa Fluor 568 (1:600)</td></tr><tr><td>Mounting medium</td><td>SouthernBiotech</td><td>00-4958-02</td><td>Fluoromount-G</td></tr><tr><td>Confocal Microscope</td><td>Olympus</td><td>NA</td><td>FV1000 confocal microscope using a 60x 1.35 numerical aperture (NA) oil objective. For vGlut1, use a 488 laser with a percentage power of 14%. For PSD-95 use a 559 laser with a percentage power of 20%.</td></tr><tr><td>Analysis software</td><td>PerkinElmer</td><td>NA</td><td>Volocity</td></tr><tr><td>Scalpel</td><td>Swann-Morton</td><td>203</td><td>No 11</td></tr><tr><td>Super Glue</td><td>Loctite</td><td>1446875</td><td></td></tr><tr><td>95% O&lt;sub&gt;2&lt;/sub&gt;, 5% CO&lt;sub&gt;2&lt;/sub&gt;</td><td>BOC</td><td>NA</td><td>Cylinder</td></tr><tr><td>24-well plate</td><td>Corning</td><td>3524</td><td></td></tr><tr><td>Glass slides</td><td>Thermo</td><td>7107</td><td>08-1.0mm thick</td></tr><tr><td>Petri Dish</td><td>Corning</td><td>430167</td><td></td></tr><tr><td>iDkk1 mice</td><td>N/A</td><td>N/A</td><td>Mice were obtained by crossing tetO Dkk1 transgenic mice with CaMKII&amp;alpha; rtTA2 transgenic mice. TetO Dkk1 transgenic mice and CaMKII&amp;alpha; rtTA2 were crossed in a heterozygous state. Both mouse lines were bred in a C57BL/6J background. Single transgenic and WT littermate were used as controls.</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents for solutions:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>NaCl</td><td>Sigma</td><td>V800372</td><td></td></tr><tr><td>KCl</td><td>Sigma</td><td>P9333</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>255793</td><td></td></tr><tr><td>KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>P9791</td><td></td></tr><tr><td>Ca&lt;sub&gt;2&lt;/sub&gt;Cl</td><td>Sigma</td><td>223506</td><td></td></tr><tr><td>Mg&lt;sub&gt;2&lt;/sub&gt;Cl</td><td>Sigma</td><td>M9272</td><td></td></tr><tr><td>NaH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sigma</td><td>71505</td><td></td></tr><tr><td>Kynurenic acid</td><td>Sigma</td><td>K3375</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma</td><td>S5761</td><td></td></tr><tr><td>Pyruvic acid</td><td>Sigma</td><td>107360</td><td></td></tr><tr><td>EDTA</td><td>Sigma</td><td>E6758</td><td></td></tr><tr><td>D-Glucose</td><td>Sigma</td><td>G5767</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S8501</td><td></td></tr><tr><td>Triton-X</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>Donkey Serum</td><td>Millipore</td><td>S30-100ml</td><td></td></tr><tr><td>PFA</td><td>Sigma</td><td>P6148</td><td></td></tr></tbody></table>

evaluation of synapse density in hippocampal rodent brain slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

Synapse loss occurs early in neurodegenerative diseases such as AD and Parkinson&#39;s disease2,11,12. However, the molecular mechanisms underlying these deficits remain poorly understood. Deficiencies in Wnt signaling have been associated with AD13,14,15,16,17. Wnts are secreted glycoproteins and play an essential role in synapse formation and the modulation of synaptic transmission18,19,20,21. Recently, we identified Wnts as key regulators of synaptic maintenance in the mature nervous system10,22. To accurately study the impact of Wnt signaling on synapses in the hippocampus of genetically modified mice, we took advantage of a brain slice preparation and confocal microscopy to evaluate changes in synapse number. We identified healthy slices in which the structure was well preserved (Figure 2A). Moreover, the selection of synaptic puncta was carefully defined, with the exclusion of large stained objects probably representing aggregated proteins (Figure 2B). This criterion was applied to all images, with the same strict analysis parameters.
We used a transgenic model system that induces the expression of the secreted Wnt antagonist Dkk1 in adult mice (iDkk1) under tetracycline control (see the Table of Materials)10,22. We demonstrated that the blockade of Wnt signaling in the adult hippocampus triggers excitatory synapse loss specifically in the CA1 stratum radiatum region (Figure 3). Figure 3A illustrates representative confocal images of excitatory pre- and post-synaptic markers (vGlut1 and PSD-95, respectively) acquired from hippocampal slices of iDkk1 mice expressing Dkk1 for two weeks, as well as their respective controls. We analyzed these images using Volocity software and demonstrated a significant reduction in the number of excitatory synapses, quantified by the co-localization of vGlut1- and PSD-95-labeled puncta in the CA1 region of the hippocampus of iDkk1 mice following the induction of Dkk1 for two weeks (Figure 3B, *p &#60;0.05; Kruskal-Wallis test; 6 mice per genotype). In the CA1 stratum radiatum, the excitatory synapse puncta number per 1,000 &#181;m3 was reduced from 500 in control mice to 300 in iDkk1 mice. In contrast, induced Dkk1 expression did not affect the number of inhibitory synapses (identified by the co-localization between the pre- and post-synaptic markers vGAT and gephyrin, respectively), as shown in Figure 4A-B (p &#62;0.05; Kruskal-Wallis test; 3 mice per genotype).
Importantly, we performed a statistical power analysis to estimate the appropriate number of slices and animals required to generate these robust results. Using R, we calculated that approximately 35 slices per condition were needed (3 images x 3 slices x 6 animals = 36) to detect a large effect size (f = 0.4) with 80% confidence (power = 0.8) in a one-way ANOVA with blocking and replication, as described in step 3.2.6.
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56153/56153fig3.jpg" /> 
Figure 3: Loss of excitatory synapses in hippocampal slices from iDkk1 mice. (A) Representative confocal images of the CA1 stratum radiatum show excitatory synapses, identified by co-localization between vGlut1 and PSD-95 (white arrows). The lower panel represents a higher magnification of the highlighted rectangle. Scale bars =&#160;2 &#181;m. (B) The percentage of excitatory synapses between control and iDkk1 was quantified using software mentioned in Table of Materials (*p &#60;0.05; Kruskal-Wallis test; 6 mice per genotype). The data are represented &#177; SEM. This figure has been modified from reference10. <a href="https://cloudflare.jove.com/files/ftp_upload/56153/56153fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56153/56153fig4.jpg" /> 
Figure 4:Inhibitory synapses are unchanged in hippocampal slices from iDkk1 mice. (A) Representative confocal images of the CA1 stratum radiatum of inhibitory synapses identified by the co-localization between vGAT and gephyrin (white arrows). The lower panel represents a higher magnification of the highlighted rectangle. Scale bars =&#160;2 &#181;m. (B) Percentage of inhibitory synapses between control and iDkk1 mice, as quantified using Volocity software (p &#62;0.05; Kruskal-Wallis test; 3 mice per genotype). The data are represented &#177; SEM. This figure has been modified from reference10. <a href="https://cloudflare.jove.com/files/ftp_upload/56153/56153fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>High Sucrose ACSF Components</td>
			<td>Concentration</td>
			<td>Notes</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>75 mM</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>25 mM</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>2.5 mM</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4, 2H2O</td>
			<td>1.25 mM</td>
			<td></td>
		</tr>
		<tr>
			<td>Kynurenic acid</td>
			<td>1.25 mM</td>
			<td></td>
		</tr>
		<tr>
			<td>Pyruvic acid</td>
			<td>2 mM</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>0.1 mM</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>1 mM</td>
			<td>Add after oxygenation of solution</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>4 mM</td>
			<td>Add after oxygenation of solution</td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>25 mM</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>100 mM</td>
			<td></td>
		</tr>
		<tr>
			<td>Submerged chamber ACSF Components</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>125 mM</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>25 mM</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>2.5 mM</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4, 2H2O</td>
			<td>1.25 mM</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>1 mM</td>
			<td>Add after oxygenation of solution</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>1 mM</td>
			<td>Add after oxygenation of solution</td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>25 mM</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: ACSF composition.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Blocking/permeabilizing Buffer</td>
			<td>Concentration</td>
			<td>Notes</td>
		</tr>
		<tr>
			<td>Donkey serum</td>
			<td>10%</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X</td>
			<td>0.50%</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>1x</td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS</td>
			<td></td>
			<td>pH to 7.4</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>1.37 M</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>27 mM</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>100 mM</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>18 mM</td>
			<td></td>
		</tr>
		<tr>
			<td>4% PFA/4% Sucrose</td>
			<td></td>
			<td>pH to 7.4</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>1x</td>
			<td>Dilute from 10x</td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>1.33 M</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>117 mM</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 2: Buffers used for immunofluorescent staining.

Watch this Scientific Journal Video about Evaluation of Synapse Density in Hippocampal Rodent Brain Slices at JoVE.com

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

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

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...

evaluation-of-synapse-density-in-hippocampal-rodent-brain-slices

Research

JoVE Journal

Neuroscience

17.0K Views.  University College London. A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

Video: Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

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

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

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

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

Measurement of Glutamate Uptake using Radiolabeled L-[3H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

Glutamate removal from the extracellular space by high-affinity Na+-dependent transporters is essential to ensure that the brain&#39;s intrinsic connectivity mechanisms work properly and homeostasis is maintained. The hippocampus is a unique brain structure that manages higher cognitive functions, and is the subject of several studies regarding neurologic diseases. The investigation of physiological and pathological mechanisms in rodent models can benefit from acute hippocampal slice (AHS) preparations. AHS has the advantage of providing reliable information on cell function since the cytoarchitecture and synaptic circuits are preserved. Although AHS preparations are commonly used in neurochemistry laboratories, it is possible to find some methodological differences in the literature. Considering that distinctive slice preparation protocols might change the hippocampal regions analyzed, this current protocol proposes a standard technique for obtaining transverse AHS from resected hippocampus. This simple-to-perform protocol may be used in mice and rats&#39; experimental models and allow several ex vivo approaches investigating neurochemical dynamics (in dorsal, intermediate and ventral hippocampus) in different backgrounds (e.g., transgenic manipulations) or after in vivo manipulations (e.g., pharmacological treatments or suitable rodent models to study clinical disorders). After dissecting the hippocampus from the rodent brain, transverse slices along the septo-temporal axis (300 &#181;m thick) were obtained. These AHS contain distinct parts of the hippocampus and were subjected to an individual neurochemical investigation (as an example: neurotransmitter transporters using their respective substrates). As the hippocampus presents a high density of excitatory synapses, and glutamate is the most important neurotransmitter in the brain, the glutamatergic system is an interesting target for in vivo observed phenomena. Thus, the current protocol provides detailed steps to explore glutamate uptake in ex vivo AHS using L-[3H]-Glutamate. Using this protocol to investigate hippocampal function may help to better understand the influence of glutamate metabolism on mechanisms of neuroprotection or neurotoxicity.

Measurement of Glutamate Uptake using Radiolabeled L-[3H]-Glutamate in Acute Transverse Slices Obtained from Rodent Resected Hippocampus

This article describes a reliable and simple way to obtain ex vivo acute hippocampal transverse slices from mice and rats using a tissue chopper. Slices obtained from resected hippocampi can be submitted for functional glutamate uptake analysis to investigate glutamatergic system homeostasis.

Glutamate removal from the extracellular space by high-affinity Na+-dependent transporters is essential to ensure that the brain&#39;s intrinsic ...

Biochemistry

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

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

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

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

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Minimizing Hypoxia in Hippocampal Slices from Adult and Aging Mice

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high fidelity. Exploration of LTP and LTD mechanisms, single neuron dendritic computation, and experience-dependent changes in circuitry, would not have been possible without this classical preparation. However, with a few exceptions, most basic research using acute hippocampal slices has been performed using slices from rodents of relatively young ages, ~P20-P40, even though synaptic and intrinsic excitability mechanisms have a long developmental tail that reaches past P60. The main appeal of using young hippocampal slices is preservation of neuronal health aided by higher tolerance to hypoxic damage. However, there is a need to understand neuronal function at more mature stages of development, further accentuated by the development of various animal models of neurodegenerative diseases that require an aging brain preparation. Here we describe a modification to an acute hippocampal slice preparation that reliably delivers healthy slices from adult and aging mouse hippocampi. The protocol&#8217;s critical steps are transcardial perfusion and cutting with ice-cold sodium-free NMDG-aSCF. Together, these steps attenuate the hypoxia-induced drop in ATP upon decapitation, as well as cytotoxic edema caused by passive sodium fluxes. We demonstrate how to cut transversal slices of hippocampus plus cortex using a vibrating microtome. Acute hippocampal slices obtained in this way are reliably healthy over many hours of recording, and are appropriate for both field-recordings and targeted patch-clamp recordings, including targeting of fluorescently labeled neurons.

This is a protocol for acute slice preparation from adult and aging mouse hippocampi that takes advantage of transcardial perfusion and slice cutting with ice-cold NMDG-aCSF to reduce hypoxic damage to the tissue. The resulting slices stay healthy over many hours, and are suitable for long-term patch-clamp and field-recordings.

Acute hippocampal slices have enabled generations of neuroscientists to explore synaptic, neuronal, and circuit properties in detail and with high ...

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

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

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

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

Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity

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

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

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

Horizontal Hippocampal Slices of the Mouse Brain

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as well as the manifestation of severe brain disorders, including Alzheimer&#8217;s disease and epilepsy. The hippocampus receives a high degree of intra- and inter-connectivity, securing a proper communication with internal and external brain structures. This connectivity is accomplished via different informational flows in the form of fiber pathways. Brain slices are a frequently used methodology when exploring neurophysiological functions of the hippocampus. Hippocampal brain slices can be used for several different applications, including electrophysiological recordings, light microscopic measurements as well as several molecular biological and histochemical techniques. Therefore, brain slices represent an ideal model system to assess protein functions, to investigate pathophysiological processes involved in neurological disorders as well as for drug discovery purposes.
There exist several different ways of slice preparations. Brain slice preparations with a vibratome allow a better preservation of the tissue structure and guarantee a sufficient oxygen supply during slicing, which present advantages over the traditional use of a tissue chopper. Moreover, different cutting planes can be applied for vibratome brain slice preparations. Here, a detailed protocol for a successful preparation of vibratome-cut horizontal hippocampal slices of mouse brains is provided. In contrast to other slice preparations, horizontal slicing allows to keep the fibers of the hippocampal input path (perforant path) in a fully intact state within a slice, which facilitates the investigation of entorhinal-hippocampal interactions. Here, we provide a thorough protocol for the dissection, extraction, and acute horizontal slicing of the murine brain, and discuss challenges and potential pitfalls of this technique. Finally, we will show some examples for the use of brain slices in further applications.

This article aims to describe a systematic protocol to obtain horizontal hippocampal brain slices in mice. The objective of this methodology is to preserve the integrity of hippocampal fiber pathways, such as the perforant path and the mossy fiber tract to assess dentate gyrus related neurological processes.

The hippocampus is a highly organized structure in the brain that is a part of the limbic system and is involved in memory formation and consolidation as ...

Preparation of Acute Slices from Dorsal Hippocampus for Whole-Cell Recording and Neuronal Reconstruction in the Dentate Gyrus of Adult Mice

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in molecular, anatomical and functional criteria suggesting a division into different sub-circuits along its rostro-caudal extent. Owing to differential connectivity and function the most fundamental distinction is made between the dorsal and the ventral hippocampus, which are preferentially involved in spatial and emotional processing, respectively. Accordingly, in vivo work regarding spatial memory formation has focused on the dorsal hippocampus.
In contrast, electro-physiological in vitro recordings have been preferentially performed on intermediate-ventral hippocampus, largely motivated by factors like slice viability and circuit integrity. To allow for direct correlation of in vivo data on spatial processing with in vitro data we have adapted previous sectioning methods to obtain highly viable transverse brain slices from the dorsal-intermediate hippocampus for long-term recordings of principal cells and interneurons in the dentate gyrus. As spatial behavior is routinely analyzed in adult mice, we have combined this transversal slicing procedure with the use of protective solutions to enhance viability of brain tissue from mature animals. We use this approach for mice of about 3 months of age. The method offers a good alternative to the coronal preparation which is frequently used for in vitro studies on dorsal hippocampus. We compare these two preparations in terms of quality of recordings and preservation of morphological features of recorded neurons.

We present a protocol to prepare acute-slices from the dorsal-intermediate hippocampus of mice. We compare this transversal preparation with coronal slicing in terms of quality of recordings and preservation of morphological features of recorded neurons.

Although the general architecture of the hippocampus is similar along its longitudinal axis, recent studies have revealed prominent differences in ...

Microscopic Analysis of Synapses in Mouse Hippocampal Slices Using Immunofluorescence

<a href='https://app.jove.com/v/56153/evaluation-of-synapse-density-in-hippocampal-rodent-brain-slices'>Source: McLeod, F., et al. Evaluation of Synapse Density in Hippocampal Rodent Brain Slices. J. Vis. Exp. (2017).</a>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 architecture through their colocalization.

Source: McLeod, F., et al. Evaluation of Synapse Density in Hippocampal Rodent Brain Slices. J. Vis. Exp. (2017).This video demonstrates a method for ...