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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/files/ftp_upload/56153/56153fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<ol>
	<li>Malenka, R. C., Bear, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LTP+and+LTD:+an+embarrassment+of+riches.">LTP and LTD: an embarrassment of riches.</a> Neuron. 44 (1), 5-21 (2004).</li><li>Arendt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+degeneration+in+Alzheimer's+disease.">Synaptic degeneration in Alzheimer's disease.</a> Acta Neuropathol. 118 (1), 167-179 (2009).</li><li>Missler, M., Sudhof, T. C., Biederer, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+cell+adhesion.">Synaptic cell adhesion.</a> Cold Spring Harb Perspect Biol. 4 (4), a005694 (2012).</li><li>Chua, J. J., Kindler, S., Boyken, J., Jahn, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+architecture+of+an+excitatory+synapse.">The architecture of an excitatory synapse.</a> J Cell Sci. 123 (Pt 6), 819-823 (2010).</li><li>Dobie, F. A., Craig, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibitory+synapse+dynamics:+coordinated+presynaptic+and+postsynaptic+mobility+and+the+major+contribution+of+recycled+vesicles+to+new+synapse+formation.">Inhibitory synapse dynamics: coordinated presynaptic and postsynaptic mobility and the major contribution of recycled vesicles to new synapse formation.</a> J Neurosci. 31 (29), 10481-10493 (2011).</li><li>Jackson, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+tau+increases+amyloid+beta+plaque+size+but+not+amyloid+beta-mediated+synapse+loss+in+a+novel+mouse+model+of+Alzheimer's+disease.">Human tau increases amyloid beta plaque size but not amyloid beta-mediated synapse loss in a novel mouse model of Alzheimer's disease.</a> Eur J Neurosci. 44 (12), 3056-3066 (2016).</li><li>Heck, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+automated+3D+detection+of+synaptic+contacts+reveals+the+formation+of+cortico-striatal+synapses+upon+cocaine+treatment+in+vivo.">A new automated 3D detection of synaptic contacts reveals the formation of cortico-striatal synapses upon cocaine treatment in vivo.</a> Brain Struct Funct. 220 (5), 2953-2966 (2015).</li><li>Sultana, R., Banks, W. A., Butterfield, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Decreased+levels+of+PSD95+and+two+associated+proteins+and+increased+levels+of+BCl2+and+caspase+3+in+hippocampus+from+subjects+with+amnestic+mild+cognitive+impairment:+Insights+into+their+potential+roles+for+loss+of+synapses+and+memory,+accumulation+of+Abeta,+and+neurodegeneration+in+a+prodromal+stage+of+Alzheimer's+disease.">Decreased levels of PSD95 and two associated proteins and increased levels of BCl2 and caspase 3 in hippocampus from subjects with amnestic mild cognitive impairment: Insights into their potential roles for loss of synapses and memory, accumulation of Abeta, and neurodegeneration in a prodromal stage of Alzheimer's disease.</a> J Neurosci Res. 88 (3), 469-477 (2010).</li><li>Harwell, C. S., Coleman, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptophysin+depletion+and+intraneuronal+Abeta+in+organotypic+hippocampal+slice+cultures+from+huAPP+transgenic+mice.">Synaptophysin depletion and intraneuronal Abeta in organotypic hippocampal slice cultures from huAPP transgenic mice.</a> Mol Neurodegener. 11 (1), 44 (2016).</li><li>Marzo, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversal+of+Synapse+Degeneration+by+Restoring+Wnt+Signaling+in+the+Adult+Hippocampus.">Reversal of Synapse Degeneration by Restoring Wnt Signaling in the Adult Hippocampus.</a> Curr Biol. 26 (19), 2551-2561 (2016).</li><li>Zoghbi, H. Y., Bear, M. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+dysfunction+in+neurodevelopmental+disorders+associated+with+autism+and+intellectual+disabilities.">Synaptic dysfunction in neurodevelopmental disorders associated with autism and intellectual disabilities.</a> Cold Spring Harb Perspect Biol. 4 (3), (2012).</li><li>Janezic, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficits+in+dopaminergic+transmission+precede+neuron+loss+and+dysfunction+in+a+new+Parkinson+model.">Deficits in dopaminergic transmission precede neuron loss and dysfunction in a new Parkinson model.</a> Proc Natl Acad Sci U S A. 110 (42), E4016-E4025 (2013).</li><li>Caricasole, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+Dickkopf-1,+a+negative+modulator+of+the+Wnt+pathway,+is+associated+with+neuronal+degeneration+in+Alzheimer's+brain.">Induction of Dickkopf-1, a negative modulator of the Wnt pathway, is associated with neuronal degeneration in Alzheimer's brain.</a> J Neurosci. 24 (26), 6021-6027 (2004).</li><li>De Ferrari, G. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Common+genetic+variation+within+the+low-density+lipoprotein+receptor-related+protein+6+and+late-onset+Alzheimer's+disease.">Common genetic variation within the low-density lipoprotein receptor-related protein 6 and late-onset Alzheimer's disease.</a> Proc Natl Acad Sci U S A. 104 (22), 9434-9439 (2007).</li><li>Liu, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficiency+in+LRP6-mediated+Wnt+signaling+contributes+to+synaptic+abnormalities+and+amyloid+pathology+in+Alzheimer's+disease.">Deficiency in LRP6-mediated Wnt signaling contributes to synaptic abnormalities and amyloid pathology in Alzheimer's disease.</a> Neuron. 84 (1), 63-77 (2014).</li><li>Purro, S. A., Dickins, E. M., Salinas, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Secreted+Wnt+Antagonist+Dickkopf-1+Is+Required+for+Amyloid+beta-Mediated+Synaptic+Loss.">The Secreted Wnt Antagonist Dickkopf-1 Is Required for Amyloid beta-Mediated Synaptic Loss.</a> J Neurosci. 32 (10), 3492-3498 (2012).</li><li>Rosi, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+Dickkopf-1+expression+in+transgenic+mouse+models+of+neurodegenerative+disease.">Increased Dickkopf-1 expression in transgenic mouse models of neurodegenerative disease.</a> J Neurochem. 112 (6), 1539-1551 (2010).</li><li>Budnik, V., Salinas, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signaling+during+synaptic+development+and+plasticity.">Wnt signaling during synaptic development and plasticity.</a> Curr Opin Neurobiol. 21 (1), 151-159 (2011).</li><li>Ciani, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signalling+tunes+neurotransmitter+release+by+directly+targeting+Synaptotagmin-1.">Wnt signalling tunes neurotransmitter release by directly targeting Synaptotagmin-1.</a> Nat Commun. 6, 8302 (2015).</li><li>Dickins, E. M., Salinas, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnts+in+action:+from+synapse+formation+to+synaptic+maintenance.">Wnts in action: from synapse formation to synaptic maintenance.</a> Front Cell Neurosci. 7, 162 (2013).</li><li>Inestrosa, N. C., Varela-Nallar, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Wnt+signalling+in+neuronal+differentiation+and+development.">Wnt signalling in neuronal differentiation and development.</a> Cell Tissue Res. 359 (1), 215-223 (2015).</li><li>Galli, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deficient+Wnt+signalling+triggers+striatal+synaptic+degeneration+and+impaired+motor+behaviour+in+adult+mice.">Deficient Wnt signalling triggers striatal synaptic degeneration and impaired motor behaviour in adult mice.</a> Nat Commun. 5, 4992 (2014).</li></ol>

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% O2, 5% CO2</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&#945; rtTA2 transgenic mice. TetO Dkk1 transgenic mice and CaMKII&#945; 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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Reagents for solutions:</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>Na2HPO4</td>
			<td>Sigma</td>
			<td>255793</td>
			<td></td>
		</tr>
		<tr>
			<td>KH2PO4</td>
			<td>Sigma</td>
			<td>P9791</td>
			<td></td>
		</tr>
		<tr>
			<td>Ca2Cl</td>
			<td>Sigma</td>
			<td>223506</td>
			<td></td>
		</tr>
		<tr>
			<td>Mg2Cl</td>
			<td>Sigma</td>
			<td>M9272</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</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>NaHCO3</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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

16.8K 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 Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

DiOLISTIC Labeling of Neurons from Rodent and Non-human Primate Brain Slices

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; Gan et al., 2000). Here we provide a detailed description of each step required together with exemplary images of good and bad outcomes that will help when setting up the technique. In our experience, a few steps proved critical for the successful application of DiOLISTICS. These considerations include the quality of the DiI-coated bullets, the extent of fixative exposure, and the concentration of detergent used in the incubation solutions. Tips and solutions for common problems are provided. This is a versatile labeling technique that can be applied to multiple animal species at a wide range of ages. Unlike other fluorescent labeling techniques that are limited to preparations from young animals or restricted to mice because they rely on the expression of a fluorescent transgene, DiOLISTIC labeling can be applied to animals of all ages, species and genotypes and it can be used in combination with immunostaining to identify a specific subpopulation of cells. Here we demonstrate the use of DiOLISTICS to label neurons in brain slices from adult mice and adult non-human primates with the purpose of quantifying dendrite branching and dendritic spine morphology.

We demonstrate the use of the gene gun to introduce fluorescent dyes, such as DiI, into neurons in brain slices from rodents and non-human primates of different ages. In this particular case, we use adult mice (3-6 months old) and adult cynomologus monkeys (9-15 years old). This technique, originally described by the laboratory of Dr. Lichtman (Gan et al., 2000), is well suited for the study of dendritic branching and dendritic spine morphology and can be combined with traditional immunostaining, if detergents are kept at a low concentration.

DiOLISTIC staining uses the gene gun to introduce fluorescent dyes, such as DiI, into neurons of brain slices (Gan et al., 2009; O'Brien and Lummis, 2007; ...

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Low-Density Primary Hippocampal Neuron Culture

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility in genetic and chemical manipulation of individual cells or defined networks. Such ease of manipulation is simpler in the reduced culture system when compared to the intact brain tissue. While many methods for the isolation and growth of these primary neurons exist, each has its own limitations. This protocol describes a method for culturing low-density and high-purity rodent embryonic hippocampal neurons on glass coverslips, which are then suspended over a monolayer of glial cells. This &#39;sandwich culture&#39; allows for exclusive long-term growth of a population of neurons while allowing for trophic support from the underlying glial monolayer. When neurons are of sufficient age or maturity level, the neuron coverslips can be flipped-out of the glial dish and used in imaging or functional assays. Neurons grown by this method typically survive for several weeks and develop extensive arbors, synaptic connections, and network properties.

This article describes the protocol for culturing low-density primary hippocampal neurons growing on glass coverslips inverted over a glial monolayer. The neuron and glial layers are separated by paraffin wax beads. The neurons grown by this method are suitable for high-resolution optical imaging and functional assays.

The ability to probe the structure and physiology of individual nerve cells in culture is crucial to the study of neurobiology, and allows for flexibility ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Recording and Modulation of Epileptiform Activity in Rodent Brain Slices Coupled to Microelectrode Arrays

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) is a promising approach when pharmacological treatment fails or neurosurgery is not recommended. Acute brain slices coupled to microelectrode arrays (MEAs) represent a valuable tool to study neuronal network interactions and their modulation by electrical stimulation. As compared to conventional extracellular recording techniques, they provide the added advantages of a greater number of observation points and a known inter-electrode distance, which allow studying the propagation path and speed of electrophysiological signals. However, tissue oxygenation may be greatly impaired during MEA recording, requiring a high perfusion rate, which comes at the cost of decreased signal-to-noise ratio and higher oscillations in the experimental temperature. Electrical stimulation further stresses the brain tissue, making it difficult to pursue prolonged recording/stimulation epochs. Moreover, electrical modulation of brain slice activity needs to target specific structures/pathways within the brain slice, requiring that electrode mapping be easily and quickly performed live during the experiment. Here, we illustrate how to perform the recording and electrical modulation of 4-aminopyridine (4AP)-induced epileptiform activity in rodent brain slices using planar MEAs. We show that the brain tissue obtained from mice outperforms rat brain tissue and is thus better suited for MEA experiments. This protocol guarantees the generation and maintenance of a stable epileptiform pattern that faithfully reproduces the electrophysiological features observed with conventional field potential recording, persists for several hours, and outlasts sustained electrical stimulation for prolonged epochs. Tissue viability throughout the experiment is achieved thanks to the use of a small-volume custom recording chamber allowing for laminar flow and quick solution exchange even at low (1 mL/min) perfusion rates. Quick MEA mapping for real-time monitoring and selection of stimulating electrodes is performed by a custom graphic user interface (GUI).

We illustrate how to perform recording and electrical modulation of 4-aminopyridine-induced epileptiform activity in rodent brain slices using microelectrode arrays. A custom recording chamber maintains tissue viability throughout prolonged experimental sessions. Live electrode mapping and selection of stimulating pairs are performed by a custom graphical user interface.

Temporal lobe epilepsy (TLE) is the most common partial complex epileptic syndrome and the least responsive to medications. Deep brain stimulation (DBS) ...

Purification of the Dendritic Filopodia-rich Fraction

Dendritic filopodia are thin and long protrusions based on the actin filament, and they extend and retract as if searching for a target axon. When the dendritic filopodia establish contact with a target axon, they begin maturing into spines, leading to the formation of a synapse. Telencephalin (TLCN) is abundantly localized in dendritic filopodia and is gradually excluded from spines. Overexpression of TLCN in cultured hippocampal neurons induces dendritic filopodia formation. We showed that telencephalin strongly binds to an extracellular matrix molecule, vitronectin. Vitronectin-coated microbeads induced phagocytic cup formation on neuronal dendrites. In the phagocytic cup, TLCN, TLCN-binding proteins such as phosphorylated Ezrin/Radixin/Moesin (phospho-ERM), and F-actin are accumulated, which suggests that components of the phagocytic cup are similar to those of dendritic filopodia. Thus, we developed a method for purifying the phagocytic cup instead of dendritic filopodia. Magnetic polystyrene beads were coated with vitronectin, which is abundantly present in the culture medium of hippocampal neurons and which induces phagocytic cup formation on neuronal dendrites. After 24 h of incubation, the phagocytic cups were mildly solubilized with detergent and collected using a magnet separator. After washing the beads, the binding proteins were eluted and analyzed by silver staining and Western blotting. In the binding fraction, TLCN and actin were abundantly present. In addition, many proteins identified from the fraction were localized to the dendritic filopodia; thus, we named the binding fraction as the dendritic filopodia-rich fraction. This article describes details regarding the purification method for the dendritic filopodia-rich fraction.

In this protocol, we introduce a method for purifying the dendritic filopodia-rich fraction from the phagocytic cup-like protrusion structure on cultured hippocampal neurons by taking advantage of the specific and strong affinity between a dendritic filopodial adhesion molecule, TLCN, and an extracellular matrix molecule, vitronectin.

Dendritic filopodia are thin and long protrusions based on the actin filament, and they extend and retract as if searching for a target axon. When the ...

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from parallel fibers (PF) to Purkinje cells (PC) is considered the basis for motor learning, and deficiencies of both LTD and motor learning are observed in various gene-manipulated animals. Common motor learning sets, such as adaptation of the optokinetic reflex (OKR), the vestibular-ocular reflex (VOR), and rotarod test were used for evaluation of motor learning ability. However, results obtained from the GluA2-carboxy terminus modified knock-in mice demonstrated normal adaptation of the VOR and the OKR, despite lacking PF-LTD. In that report, induction of LTD was only attempted using one type of stimulation protocol at room temperature. Thus, conditions to induce cerebellar LTD were explored in the same knock-in mutants using various protocols at near physiological temperature. Finally, we found stimulation protocols, by which LTD could be induced in these gene-manipulated mice. In this study, a set of protocols are proposed to evaluate LTD-induction, which will more accurately allow examination of the causal relationship between LTD and motor learning. In conclusion, experimental conditions are crucial when evaluating LTD in gene-manipulated mice.

In some gene-manipulated animals, using a single protocol may fail to induce LTD in cerebellar Purkinje cells, and there may be a discrepancy between LTD and motor learning. Multiple protocols are necessary to assess LTD-induction in gene-manipulated animals. Standard protocols are shown.

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...