This protocol presents a robust, detailed method to obtain highly pure synaptosomes, synaptic vesicles, and other synaptic fractions from the mouse brain. This method enables the evaluation of synaptic processes, including the biochemical analysis of protein localization and function with compartmental resolution.
Synaptic terminals are the primary sites of neuronal communication. Synaptic dysfunction is a hallmark of many neuropsychiatric and neurological disorders. The characterization of synaptic sub-compartments by biochemical isolation is, therefore, a powerful method to elucidate the molecular bases of synaptic processes, both in health and disease. This protocol describes the isolation of synaptic terminals and synaptic sub-compartments from mouse brains by subcellular fractionation. First, sealed synaptic terminal structures, known as synaptosomes, are isolated following brain tissue homogenization. Synaptosomes are neuronal pre- and post-synaptic compartments with pinched-off and sealed membranes. These structures retain a metabolically active state and are valuable for studying synaptic structure and function. The synaptosomes are then subjected to hypotonic lysis and ultracentrifugation to obtain synaptic sub-compartments enriched for synaptic vesicles, synaptic cytosol, and synaptic plasma membrane. Fraction purity is confirmed by electron microscopy and biochemical enrichment analysis for proteins specific to sub-synaptic compartments. The presented method is a straightforward and valuable tool for studying the structural and functional characteristics of the synapse and the molecular etiology of various brain disorders.
Synapses are the basic computational units of the brain through which neurons communicate and exert diverse and exquisitely complex functions. Synapses are, thus, fundamental to the health of the brain1; synaptic dysfunction is implicated as a source or result of many disorders2. Synapses are constituted by pre- and post-synaptic terminals, extensions of two different neurons that are closely apposed and separated by a synaptic cleft traversed by synaptic adhesion molecules. Information flows from the pre- to post-synaptic compartment in the form of chemical messengers called neurotransmitters1. The molecular processes involved in neurotransmission are active areas of research3,4,5. Understanding the pathogenic processes within synaptic terminals and the response of synapses to pathology in other neuronal sub-compartments are crucial steps to addressing disorders of the brain1,2. Several methodological advancements, predominantly applied to murine models, have advanced this pursuit6. The isolation of synaptic fractions by differential centrifugation is one such paradigm-shifting method that has enabled the detailed evaluation of synaptic processes in health and disease.
The adult human brain consists of 80-90 billion neurons7,8. Among murine species, the rat brain contains approximately ~200 million neurons, while mice have ~70 million9,10. Each neuron forms thousands of specific synaptic connections with a network of highly polarized neurons intermingled with glial cells and dense vasculature. In such complex and heterogeneous tissue, it was once unthinkable to isolate and study synapses as an independent system. In the 1960s, Victor Whittaker, Catherine Hebb, and others made this possible by isolating intact synaptic terminals using subcellular fractionation11,12,13,14. In an attempt to isolate synaptic vesicles (SVs), they homogenized brains through liquid shear force in iso-osmotic (0.32 M) sucrose followed by ultracentrifugation. They obtained pinched-off, plasma membrane-enclosed, intact nerve terminals or varicosities, which they called nerve-ending particles (NEPs)11,13. As the structural and functional characteristics of the synapse were preserved in these structures, NEPs were later termed "synaptosomes" for congruence with other subcellular organelles13,15. It is worth noting that the work of Eduardo de Robertis and colleagues, who coined the term "synaptic vesicle", overlapped with that of Whittaker and colleagues and contributed to the validation of "synaptosome" isolation and characterization16,17,18.
Synaptosomes are physiologically active structures that contain all the cellular and molecular properties required for the storage, release, and reuptake of neurotransmitters13,18. The preservation of key synaptic characteristics in vitro and freedom from non-synaptic components also contribute to the utility of this isolation method. Synaptosomes have contributed immensely to the understanding of the chemical and physiological properties of neurotransmission and are now being used to study synaptic molecular processes and their alterations in disease19,20,21,22,23. Synaptosomes are also the initial source material for isolating synaptic components such as SVs, clathrin-coated vesicles (CCVs), synaptic cytosol, synaptic plasma membrane, synaptic mitochondria, synaptic adhesion molecules, and other components of interest, which can facilitate the understanding of the molecular mechanisms of synaptic function18,19,20,24,25,26,27,28. These sub-synaptic components can be obtained by the osmotic lysis of synaptosomes and sucrose density gradient ultracentrifugation15,29. Although the original subcellular fractionation method by Whittaker's research group is known to be efficient in isolating quality synaptosomes and SVs13,30, recent optimizations enhance the purity of the subcellular fractions22,23,31,32. This article provides a highly detailed and accessible version of a classic protocol for the subcellular fractionation of murine brain tissue to isolate synaptosomes, SVs, and other sub-synaptic components.
All experiments with mice were approved by the Institutional Animal Care and Use Committee (IACUC) at Yale University (Protocol 2021-11117) and performed in a facility accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC). Animal care and housing complied with the Guide for the Care and Use of Laboratory Animals33 and were provided by the Yale Animal Resource Center (YARC). Animals were maintained in a 12 h light/dark cycle with ad libitum access to food and water. Five to eight mice or two to four rats per genotype or condition are required for the following protocol. Fewer rats are necessary due to their larger brain volumes. Similarly, the age of the experimental animals may affect fraction yield; additional mice may be required for ages less than 2 months. Otherwise, the outlined procedures apply to both murine species and healthy adult animals of any age. The representative data presented in this study utilized wild-type (C57BL/6J) mice (age = 2 months; four males and four females per replicate) obtained from a commercial source (see Table of Materials).
1. Experimental preparation
NOTE: This protocol requires ~11 h for a single researcher to complete. It is highly recommended to complete benchtop setup (Figure 1), buffer preparation (Table 1), the precooling of centrifuges and rotors to 4 °C, and the collection and labeling of necessary materials and equipment (see Table of Materials) the day prior to protocol execution, where applicable.
Figure 1: Benchtop setup. Prior to brain dissections, (A) Dounce glass homogenizers and (B) all buffers were chilled on ice. (C) Protease inhibitor stock solutions were thawed on ice. A second container of wet ice for centrifuge tubes, a Dewar of liquid nitrogen (not shown), and (D) a container of dry ice for short-term storage of the samples flash-frozen in liquid nitrogen were obtained. (E) Microcentrifuge tubes were pre-labeled for all samples, as four aliquots of each subcellular fraction sample per genotype or condition were collected during this procedure (time-saving tip: thoroughly label all the tubes the day before the experiment is performed). (F) An appropriate biohazard waste container, (G) 70% ethanol, (H) surgical tools, and (I) an absorbent surface pad. The required centrifuge tubes and disposables were set aside for efficient access during protocol implementation (not shown). Please click here to view a larger version of this figure.
Table 1: Composition of the subcellular fractionation buffers. Please click here to download this Table.
Figure 2: Overview of the subcellular fractionation protocol. Summary schematic of the subcellular fractionation steps and collected samples. Please click here to view a larger version of this figure.
2. Mouse brain excision
Figure 3: Craniofacial anatomy. (A) Dorsal view of a mouse skull with relevant cranial structures indicated. (B) Left lateral view of a mouse skull and brain with relevant cranial structures and anatomical directions indicated. The dashed lines represent the locations where incisions should be made. Please click here to view a larger version of this figure.
3. Synaptosome preparation
NOTE: The schematics of this procedure are shown in Figure 4.
Figure 4: Synaptosome preparation. Schematic of step 3, the generation of synaptosomes (P2'). Please click here to view a larger version of this figure.
4. Hypotonic lysis
NOTE: The schematics of this procedure are shown in Figure 5.
Figure 5: Hypotonic lysis. Schematic of step 4, the hypotonic lysis of synaptosomes to generate the lysis supernatant (LS1) and synaptosomal membrane fractions (LP1). Please click here to view a larger version of this figure.
5. Synaptic vesicle isolation
NOTE: The schematics of this procedure are shown in Figure 6.
Figure 6: Synaptic vesicle isolation and synaptic plasma membrane isolation. (A) Schematic of step 5, the isolation of synaptic cytosol (LS2) and synaptic vesicle (LP2) fractions, and (B) step 6, the generation of myelin (MF), synaptic plasma membrane (SPM), and mitochondrial (Mito.) fractions following the ultracentrifugation of sucrose gradients. Please click here to view a larger version of this figure.
6. Synaptic plasma membrane isolation
The presented method results in 11 brain subcellular fractions that can be subjected to further purification and various forms of downstream analysis35,36. The gold standard method to assess the quality of synaptosomes, SVs23, and other components is electron microscopy (EM) (Figure 7). Quantitative immunoblotting for proteins that are present in specific subcellular fractions can also be performed to assess markers of fraction purity (Figure 8). For example, immunoblot analysis of fractions reveals the enrichment of N-cadherin (CDH2, UniProt name) in the synaptic plasma membrane fraction (SPM), α-synuclein (SYUA) in the synaptic cytosol (LS2), synaptophysin (SYPH) in the synaptic vesicle fraction (LP2), and myelin basic protein (MBP) in the myelin fraction (MF) when compared to protein levels in the initial whole brain homogenate (Total) (Figure 8). Once fraction purity has been established (for example, note the absence of CDH2 in the LS2 fraction or the many-fold increase in SYPH in the LP2 fraction), quantitative immunoblotting can be used to determine the localization of proteins of interest or query differences in protein distribution between genotypes or treatments. Understanding the subcellular localization of synaptic proteins can enable the dissection of previously undescribed protein functions. Further, this method may elucidate trafficking defects or synaptic dysfunction in disease states, especially when paired with functional assays. For example, our team has used this method to identify a pool of enzymatically active palmitoyl protein thioesterase 1 that is enriched in the synaptic cytosol19.
Figure 7: Electron microscopy (EM) of synaptosomes. (A) Representative EM image of a synaptosome containing synaptic vesicles (arrow). (B) Representative EM image of a synaptosome with both pre- (arrow) and post-synaptic components (double arrow). (C) Representative EM image of a synaptosome containing synaptic vesicles and a mitochondrion (arrow) (scale bars = 100 nm). Please click here to view a larger version of this figure.
Figure 8: Immunoblot analysis of subcellular fractions. (A) Markers of subcellular fraction purity (indicated with UniProt nomenclature) are appropriately localized compared to the whole brain homogenate (total): N-cadherin (CDH2) in the synaptic plasma membrane fraction (SPM), synaptophysin 1 (SYPH) and synaptojanin 1 (SYNJ1) in the synaptic vesicle enriched fraction (LP2), α-synuclein (SYUA) in the synaptic cytosol (LS2), and myelin basic protein (MBP) in the myelin fraction (MF). (B) Immunoblot quantification analysis reveals the enrichment (fold-change from total) of fraction purity markers. Data are represented as mean ± standard deviation on a log10 scale. The dotted line indicates a 1.5-fold change (y = 0.176) (n = 3 replicate experiments with 8 wild-type mice; age = 2 months; n = 4-5 blots for SYPH, SYUA, MBP, with n = 3 plotted values previously published by Gorenberg et al.19; n = 5 for SYNJ1; n = 1 for CDH2). Please click here to view a larger version of this figure.
In their seminal studies, Whittaker and colleagues used four morphological criteria to identify synaptosomes: (1) the structures have a sealed plasma membrane; (2) the structures contain SVs resembling those in nerve terminals and varicosities in situ in size and number; (3) the structures possess one or more small mitochondria; and (4) the presynaptic membrane is frequently adhered to a post-synaptic component11,12,13. Though the first two criteria generally apply to every isolation method, in the most recent protocols described in this article, not all resulting synaptosomes will have mitochondria and attached post-synaptic terminals. Approximately 60% of the synaptosomes will have mitochondria, and only up to 15% are estimated to have attached post-synaptic terminals37. If post-synaptic components are of particular interest, the use of an isotonic Krebs-like homogenization buffer and pressure filtration for enrichment are known to yield high concentrations of synaptosomes with post-synaptic terminals (also termed synaptoneurosomes)22,38.
The method of sacrificing the animal can impact the quality of synaptosomes and synaptic subfractions. Adult animals sacrificed using a euthanasia method that does not require anesthesia will result in the best fraction quality. Further, the brains should be freshly dissected, not frozen, and homogenized using a 1:10 ratio of homogenization buffer (weight/volume) for the most viable synaptic fractions22. The brain has a heterogeneous population of synapses that can be differentiated by the type of neurotransmitters they carry. Synaptosome formation is generally unaffected by synapse type or neurotransmitter content13. An exception is mossy fibers in the cerebellum, which are known to be disrupted in optimal conditions for obtaining synaptosomes from the rest of the brain39,40. Thus, removal of the cerebellum prior to brain homogenization is recommended if the exclusion of this region does not affect the experimental goal. If interested in isolating synaptosomes of a particular neurotransmitter character, areas of the brain that are enriched for neurons containing the neurotransmitter of interest can first be isolated. However, this approach will impose limitations on the final fraction yield, depending on the size of the region of interest (the age of animals is also, therefore, a consideration). There are immunochemical methods for the isolation of neurotransmitter-specific synaptosomes, but the viability and yield will be significantly compromised22. If assessing synaptosome metabolic viability is important, the measurement of neurotransmitter release41,42 or certain enzymatic assays43 can be employed.
Common contaminants in synaptosome preparations include microsomes, free mitochondria, SVs, and neuronal and glial membranes. Contamination can be reduced by increasing the number of washes at the P1 and P2 fractions22 and avoiding resuspension of the red mitochondrial pellet in subsequent steps. In experiments where metabolic viability and time are crucial, reducing the number of washes and using Ficoll or Percoll gradients over sucrose gradients will be helpful44,45,46. These methods also reduce contamination significantly. Whittaker's original protocol yielded high-quality SVs. Further optimization by Nagy et al.23, included in this method, produces SVs with remarkable homogeneity and purity without compromising significantly on yield36. If specific SV subtypes are of interest, such as glutamatergic (VGLUT-1-containing) or GABAergic (VGAT-1-containing) SVs, immunoisolation using specific antibodies can be performed47,48. Alternative methods are also available for isolating CCVs from synaptosomes, which, due to differential density, may not be present at the same interface as SVs obtained with this method20,49,50.
Overall, the present protocol to isolate synaptic components can be further optimized to obtain fractions with improved homogeneity and viability based on the quality and quantity of the source brain tissue and the experimental goals. For further troubleshooting details, one should refer to book chapters by Dunkley and Robinson22 and Ganzella et al.36.
The authors have nothing to disclose.
We would like to thank P. Colosi for EM image preparation. This work was supported by the National Institutes of Health (R01 NS064963, SSC; R01 NS110354, SSC; R01 NS083846, SSC; R21 NS094971, SSC; T32 NS007224, SMT; T32 NS041228, SMT), the United States Department of Defense (W81XWH-17-1-0564, SSC; W81XWH-19-1-0264, VDJ), Aligning Science Across Parkinson's (ASAP) Collaborative Research Network (SSC), and the Michael J. Fox Foundation Target Advancement Program (MJFF-020160, SSC & VDJ). We created graphical illustrations using BioRender.com.
1 mL TB Syringe | BD | 309649 | |
1.5 mL Eppendorf Tubes | USA Scientific | 1415-2500 | |
14 mL, Open-Top Thinwall Ultra-Clear Tube | Beckman Coulter | 344060 | Compatible with SW 40 Ti |
23 Gauge Precision Glide Hypodermic Needle | BD | 305145 | |
26.3 mL, Polycarbonate Bottle with Cap Assembly | Beckman Coulter | 355618 | Compatible with Ti70 |
3.5 mL, Open-Top Thickwall Polypropylene Tube | Beckman Coulter | 349623 | Compatible with TLA-100.3 |
50 mL Falcon Tubes | Fisher Scientific | 14-432-22 | |
Amicon Ultra-15 Centrifugal Filter Unit | Millipore Sigma | UFC901024 | |
Aprotinin | Sigma-Aldrich | A6279 | 1 mg/mL in diH2O |
Avanti J-26 XP Centrifuge | Beckman Coulter | B22984 | <26,000 rpm |
Benchtop HDPE Dewar Flask | Thermo Scientific | 5028U19 | |
C57BL/6J Mice | The Jackson Labs | 000664 | |
Centrifuge 5810R | Eppendorf | EP022628168 | <14,000 rpm |
complete, Mini, EDTA-free Protease Inhibitor Cocktail Tablets | Roche | 11873580001 | Add 1 tablet per 50 mL of solution |
Curved Forceps | Fine Science Tools | 11273-20 | |
Fine Surgical Scissors | Fine Science Tools | 8r | |
Glas-Col Tissue Homogenizing System | Cole-Parmer | UX-04369-15 | |
Graefe Forceps | Fine Science Tools | 11650-10 | |
High-Speed Polycarbonate Round Bottom Centrifuge Tubes | ThermoFisher | 3117-0500 | Compatible with JA20 |
Isofluorane | Henry Schein Animal Health | NDC 11695-6776-2 | |
JA-20 Rotor | Beckman Coulter | 334831 | |
Leupeptin | American Bio | AB01108 | 1 mg/mL in diH2O |
N-[2-Hydroxyethyl] piperazine-N’-[2-ethanesulfonic acid] (HEPES) | American Bio | AB00892 | |
Optima L-80 XP Ultracentrifuge | Beckman Coulter | <100,000 rpm | |
Optima TLX Ultracentrifuge | Beckman Coulter | <120,000 rpm | |
Pepstatin A | Thermo Scientific | 78436 | 1 mg/mL in DMSO |
Phenylmethylsulfonyl fluoride (PMSF) | American Bio | AB01620 | |
Pierce BCA Protein Assay Kit | Thermo Scientific | 23335 | For determination of protein concentration |
Pipette Tips | |||
Serological Pipettes | |||
Sucrose | Sigma-Aldrich | S0389 | |
Surgical Scissors | Fine Science Tools | 14002-12 | |
SW 40 Ti Swinging-Bucket Rotor | Beckman Coulter | 331301 | |
Teflon-Coated Pestle and Mortar Tissue Grinder | Thomas Scientific | 3431D94 | |
Ti70 Rotor | Beckman Coulter | 337922 | |
TLA-100.3 Rotor | Beckman Coulter | 349490 | |
Tube Revolver | Dot Scientific | DTR-02VS |