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

Summary

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.

Abstract

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.

Protocol

Solutions to Prepare:

1. Antibody Buffer:
   • 150 mM NaCl
   • 50 mM Tris-Base (Fisher, Cat. No: BP152-5, 50 mM) - 1.21 g
   • 1% BSA (Sigma, Cat. No: A2153, 1%) - 2.0 g
   • 100 mM _L-lysine (Sigma, Cat. No: L-1137, 100 mM) - 3.65 g
   • Adjust pH to 7.4
   • 0.04% Azide
   • Adjust volume to 200 ml with distilled H₂O.
   • Filter through 0.22μm filter (Millipore, Cat. No: SCGPU02RE).
2. PFA Diluant:
   ....

Discussion

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,.......

Materials

Name	Company	Catalog Number	Comments
	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

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