This protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers.
One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become imperative to develop objective approaches to quantify changes in synaptic connectivity. Starting from sample fixation, this protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers. The number of these colocalizations is quantified using a plug in Puncta Analyzer (written by Bary Wark, available upon request, c.eroglu@cellbio.duke.edu) under the ImageJ analysis software platform. The synapse assay described in this protocol can be applied to any neural tissue or culture preparation for which you have selective pre- and postsynaptic markers. This synapse assay is a valuable tool that can be widely utilized in the study of synaptic development.
Solutions to Prepare:
Preparation of Neuronal Cultures:
The protocol described here is applicable to any primary neuronal cultures grown on 12 mm glass coverslips (Karl Hecht, No. O, Cat. No: 99010) in 24-well plates (Falcon, 35-3047). For example, in our laboratory we culture rat retinal ganglion cells (RGCs) purified from rat retina harvested from P5-7 animals1,2. Cells are grown on glass coverslips coated with poly-d-lysine (Sigma, Cat. No: P6407) and mouse laminin (Cultrex, Cat. No: 3400-010-01). We utilize this culture preparation in a couple of different ways for our synapse assay. One manipulation that we perform involves culturing RGCs either in the presence or absence of astrocyte-secreted synaptogenic factors. Alternatively, we also employ these different treatment conditions in experiments where RGCs have been transfected to overexpress a protein of interest. In the latter case, we co-transfect the cells with a cell label (e.g. GFP or tdTomato). These different experimental approaches affect how one performs certain steps of a synapse assay, which we clarify below.
1. Fixing Dissociated Purified RGCs
2. Blocking Unspecific Binding Sites on the RGCs
3. Applying Primary Antibody Solution
4. Applying Secondary Antibody Solution
5. Mounting Coverslips
6. Imaging
For imaging, a fluorescence microscope equipped with a camera capable of taking pictures at 4 different channels is necessary to be able to image both synaptic markers, your cell fill and nuclei (DAPI/optional). Cells should be imaged using an oil immersion 63x objective. We image using the Zeiss AxioImager fluorescence microscope with the Zeiss Plan-APOCHROMAT 63x/1.4 Oil DIC ∞/0.17 objective.
7. Image Analysis and Co-localized Puncta Quantification
BRAIN SECTIONS:
The synapse assay can be applied to cryosections from brain, and to any other nervous system tissue (such as spinal cord or retina) provided that there is a suitable pre- and postsynaptic marker pair (with antibodies that work well in sections) that can be utilized to identify the synapses you wish to quantify. The synapse assay can reveal the temporal regulation of synapse formation in a given brain region and can quantify effects on synaptic connectivity in transgenic animals or in a sample that has been manipulated in some other fashion.
1. Harvesting Brain Tissue from Mice
All animal procedures should be done in concordance with IACUC animal protocols.
2. Fixation
3. Embedding/Cryosectioning
4. Blocking Sections
5. Applying Primary Antibodies
6. Applying Secondary Antibodies
7. Mounting
8. Imaging
IMPORTANT: A confocal microscope with at least 3 channels is required for the imaging described here. We image on a Leica SP5 laser-scanning confocal microscope using a 63x oil immersion objective.
9. Image Quantification
Keys to Success:
Purified RGCs:
Brain Sections:
Representative Results:
The synapse assay described above is designed to capture changes in synaptic connectivity in vitro and in vivo. In our lab, we utilize the synapse assay to determine the effect of either individual or multiple astrocyte-secreted molecules on synapse formation. We commonly perform this synapse assay on purified RGCs that we culture in vitro.
A well described effect of chronic application of astrocyte-conditioned media (ACM) in purified RGC cultures is a multiple-fold increase in the number of synapses that are formed between RGCs3,4,5,6. Figure 1A and 1B show representative images of untransfected purified RGCs treated with either basal growth media or with ACM. After staining for excitatory pre- and postsynaptic markers, the ACM-induced increase in synapse formation is qualitatively evident (Figure 1A, 1B). Indeed, this finding has been corroborated by several studies that have used electrophysiology to show that ACM-induced synapses are functional and electron microscopy to show that the synapses are ultrastructurally normal3,4,6. Other work in our laboratory has identified the calcium channel subunit α2δ-1 as the neuronal receptor for a strongly synaptogenic astrocyte-secreted molecule, thrombospondin3. Here we include the results of an experiment in purified RGCs that was performed to determine whether increased levels of α2δ-1 further enhances ACM-induced synapse formation (Figure 3).
RGCs were cotransfected with either an empty vector or with a construct encoding α2δ-1 with a separate construct encoding tdTomato after 5 days in vitro (DIV5). Following chronic treatment with either ACM or GM, RGCs were fixed and stained on DIV 11. Following immunolabeling of pre- and postsynaptic markers of excitatory synapses we see a qualitatively obvious increase in synapse number in purified RGCs expressing either an empty vector (pcDNA3.1) or α2δ-1 (Fig 1C, 1D). We quantify the synaptogenic effect of ACM using Puncta analyzer to count synapse number (Fig 2) for at least 15 neurons from each condition. A sample of this size allows us to calculate the average number of synapses per neuron and find a statistically significant, ~10-fold increase in synapses formed by ACM-treated neurons that are expressing an empty vector (Fig 3, gray bars). Furthermore, we show that overexpression of α2δ-1 leads to a significant potentiation of ACM-induced synapse formation (~20-fold. Fig 3, black bars).
In addition to cultured neurons, we can quantify synaptic density in different brain regions using this technique. The superior colliculus is a brain structure that receives retinocollicular projections, originating from RGCs in the retina7,8,9. Over postnatal development, the number of excitatory synapses formed by RGCs onto their targets in the superior colliculus dramatically increases from P7 to P217,8,9.
Using our quantification technique, we show that the number of retinocollicular synapses formed in the superior colliculus increases from P7 to P14. To do this, we quantified synaptic density at P7 and again at P14. We stain the superior colliculus for PSD-95 (postsynaptic) and for VGlut2 (presynaptic marker specific to RGC synapses in the superior colliculus). The outer synaptic region of the superior colliculus was imaged (Fig 4A) and co-localized VGlut2/PSD-95 synapses were quantified (Fig 4B, 4C) from at least 3 sections per animal and from at least 3 animals per time point. Quantification of the resulting data clearly demonstrates an over three-fold significant increase in the number of synapses between P7 and P14. These results are in agreement with previously published findings utilizing electron microscopy (Fig 4D)9.
In conclusion, the method of synapse number quantification we describe here is a useful tool to determine synapse number and density in culture and in neural tissues, enabling us to study the effects of manipulating synapse formation in vitro or in vivo.
Figure 1: Representative synaptic staining in purified RGCs. (A and B) 3 days in vitro (DIV) RGCs were cultured either in (A) basal growth media (GM) or in (B) pro-synaptogenic mouse astrocyte conditioned media (ACM) for an additional 6 days. The cells were then labeled for bassoon (presynaptic, red) and homer (postsynaptic, green). Mouse ACM strongly stimulates synapse formation between RGCs as determined by the increase in the number of co-localized bassoon and homer puncta. (C and D) 5DIV RGCs were transfected with a plasmid to overexpress the calcium channel subunit α2δ-1. Transfected cells were identified with a tdTomato cell fill (blue) and have been stained for presynaptic marker synapsin and postsynaptic marker homer. Arrows indicate synapses. Scale bar represents 20 μm.
Figure 2: Quantification of synapse number using Puncta Analyzer. Shown here is an example of (a) an original image of a purified retinal ganglion cell overexpressing the thrombospondin receptor α2δ-1 and is stained for presynaptic marker synapsin and postsynaptic marker homer. (b) Images corresponding to the masks created in each channel when analyzing puncta in Puncta Analyzer. (c) Puncta Analzyer will create an image such as the one shown here in which puncta are indicated by small black dots (inset). Also shown is (d) the numerical output of the application where puncta number along with several other parameters are provided in textual/numerical form (bold text added for emphasis).
Figure 3: Representative result for synapse assay. Shown here is the synaptogenic effect of chronic treatment of RGCs with astrocyte-conditioned media (ACM) in transfected RGCs expressing either empty vector (pcDNA3.1, gray bars) or the thrombospondin receptor α2δ-1. Error bars represent the SEM. GM = Growth Media. ACM = (Rat) Astrocyte Contitioned Media.
Figure 4: Quantification of synaptic density in mouse superior colliculus. (a) To determine the developmental changes in the number of glutamatergic synapses established by RGCs onto its superior collicular targets in the rodent brain, we stained cryosections from mouse brain with antibodies against the presynaptic marker VGlut2 (green) and the postsynaptic marker, PSD-95, (red). We imaged the outer 150 x 150 μm region of the mouse superior colliculus (SC) corresponding to the synaptic target region for RGCs by using a laser scanning confocal microscope. A z-stack for each SC section was collected for a total depth of 5 μm (15 x 0.33 μm optical sections). Maximum image projections (MIPs) were generated for groups of 3 consecutive optical sections yielding 5 MIPs/section each representing 1 μm of depth. Shown below is a representative MIP taken from superior colliculus of a P14 WT mouse. The presynaptic marker, VGlut2, is shown in green and the postsynaptic marker, PSD-95, is shown in red. (c) Numerical output produced by Puncta Analyzer. (d) Quantification of the analysis of synapse number for at least 3 sections per animal with 3 animals per time point (error bars represent SEM). Scale bar represents 50 μm.
The synapse assay described above is based in the context of our experimental goals, wherein we focus largely on excitatory projections of RGCs, either in purified culture or in brain section. We have provided a reference table listing antibodies that work well for labeling excitatory synapses (Table 1).
This synapse assay can be adapted to quantify synapse number of any neuronal population or any other synaptic subtype for which there is a selective pre- and postsynaptic marker. For example, the synapse assay described here can be applied to studying GABA-ergic (as opposed to glutamatergic) synapses by using appropriate pre- and postsynaptic markers10,11.
This synapse assay is an important tool for labs investigating changes in synaptic connectivity between neurons. While it is necessary to corroborate findings of this assay with electron microscopy and electrophysiology, it nevertheless offers a relatively easy way to answer important questions about the molecular cues that modulate a neurons’ ability to form synapses.
The authors have nothing to disclose.
Puncta Analyzer Plug-in for Image J was written by Barry Wark (current address: Physion Consulting) in the lab of Ben A. Barres (Stanford University).
Funding;
Antigen | Species | Monoclonal/Polyclonal | Vendor | Catalog Number | Dilution | Works in Culture | Works in Sections | |
Presynaptic | Synapsin | Rabbit | Polyclonal | Synaptic Systems | 106004 | 1:750 | Y | N.D. |
Synapsin | Mouse | Monoclonal | Synaptic Systems | 106001 | 1:500 | Y | Y | |
Bassoon | Mouse | Monoclonal | Assay Designs | VAM-PS003F | 1:500 | Y | Y | |
Bassoon | Guinea Pig | Polyclonal | Synaptic Systems | 141004 | 1:1000 | Y | N.D. | |
Synaptotagmin 1 | Rabbit | Polyclonal | Synaptic Systems | 105002 | 1:750 | Y | N | |
Synaptobrevin 2 (C1.69.1) |
Mouse | Monoclonal | Synaptic Systems | 104211 | 1:500 | Y | Y | |
Synaptophysin (C1.7.2) |
Mouse | Monoclonal | Synaptic Systems | 101011 | 1:500 | Y | Y | |
VGlut1 | Mouse | Monoclonal | Millipore | MAB5502 | 1:2500 | N | Y | |
VGlut1 | Guinea pig | Polyclonal | Millipore | AB5905 | 1:2500 | N | Y | |
VGlut2 | Guinea pig | Polyclonal | Millipore | AB2251 | 1:2500 | N | Y | |
Postsynaptic | PSD-95 (6G6-1C9 clone) |
Mouse | Monoclonal | Affinity Bio Reagents | MA1-045 | 1:750 | Y | N |
PSD-95 | Rabbit | Polyclonal | Zymed | 51-6900 | 1:500 | N | Y | |
Homer | Mouse | Monoclonal | Synaptic Systems | 160011 | 1:500 | Y | N.D. | |
Homer | Rat | Polyclonal | Millipore | AB5875 | 1:500 | Y | Y | |
Gephyrin | Rabbit | Polyclonal | Synaptic Systems | 147003 | 1:500 | Y | Y | |
Gephyrin | Mouse | Monoclonal | Synaptic Systems | 147 | 1:200 | Y | Y |
Table 1: Lists examples of good pre- and postsynaptic markers that we have successfully utilized in our synapse assay. Be aware that this is not an exhaustive list of all available markers. Y = Yes, N = No, N.D. = Not Determined.
Reagent | Company | Cat. No. |
PBS | Invitrogen | 20012-027 |
poly-d-lysine | Sigma | P6407 |
Laminin | Cultrex | 3400-010-01 |
Triton X-100 | Roche Diagnostics Gmbh | 9002-93-1 |
Normal Goat Serum | Gibco | 16210 |
VectaShield with DAPI | Vector Laboratories | H-1200 |
OCT | Tissue-Tek | 4583 |
Tris-Base (50 mM) | Fisher | BP152-5 |
Bovine Serum Albumin | Sigma | A2153 |
l-lysine | Sigma | L-1137 |
16% PFA solution | Electron Microscopy Sciences | 15711 |
Granular PFA | Electron Microscopy Sciences | 19210 |
24-well culture plate | Falcon | 35-3047 |
Goat anti-mouse Alexa conjugated antibodies | Invitrogen | — |
Supply | Company | Cat. No. |
12mm, No. 0 glass coverslips | Karl Hecht Gmbh | 1105209 |
No. 1.5 glass coverslip (for slices) | VWR Scientific | 48393241 |
Glass slides | VWR Scientific | 48311-703 |