synaptosomes are enriched in neuron-specific and depleted in glia-specific proteins
4.2 FASS in FM-mode enriches intact, single VGLUT1 VENUS synaptosomes
Previously the light scattering of synaptosomes had been reported to be in between the signal of 0.75 and 1.5 µm polystyrene microspheres (Gylys et al., 2004a; Wolf and Kapatos, 1989b). My results indicate that particles in this size range are not single synaptosomes, but rather correspond to aggregates. These findings are in conflict with the previous studies by Wolf and colleagues and Gylys and colleagues, but are supported by that fact that in the present study: (i) VGLUT1VENUS positive particles isolated in the size range of 0.75-1.5 µm were also enriched in VIAAT and PLP as analyzed by Western blotting.
(ii) Staining with FM4-64 revealed that a large number of particles in the synaptosomal preparation fall in a size range below 0.75 µm and VGLUT1VENUS positive particles were also present among these particles. (iii) Western blotting, immunofluorescence microscopy and electron microscopy all show that sorted VGLUT1VENUS
4.2.1 Evidence from flow cytometry
positive particles that fell into the size range below 0.75 µm represent intact single synaptosomes.
During flow analysis and sorting, particles are exposed to large pressure differentials and shearing forces that occur when the fluid stream that carries the particles exits the pressurized fluidics system through a 70 µm size nozzle. If particles are unaltered by the passage through the sort-instrument one expects that they will present the same apparent light-scattering characteristics as unsorted particles. In my FSC-triggered experiments I observed a reduction in the fraction of large and fluorescently positive particles. This can be
considered as an indication for the breakdown of pre-existing aggregates during the sort process. Several weakly fluorescent or autofluorescent particles would result in an overall positive fluorescence of an aggregate, which if broken down, generates a number of smaller non-fluorescent particles. In addition, the presence of a single positive synaptosome will render an aggregate of otherwise negative elements fluorescently positive. In line with our observation, breakdown of these aggregates results in a systematic reduction of particle sizes, but not of the overall fluorescence intensity.
Alternatively the reduction in large particles could result from the disintegration of single fluorescent synaptosomes by the same shearing forces and pressure differentials.
Indeed I experienced this phenomenon when testing high pressure/speed sorting on an alternative instrument (MofloXDP from BeckmanCoulter, Data not shown). As a result I could see a major drop in fluorescence intensities consistent with the loss of VGLUT1VENUS vesicles from the synaptosomes. Contrary to this, the fluorescence intensity of isolated VGLUT1VENUS
Finally, FM4-64 staining revealed a large pool of membrane containing particles in a smaller size range among which a population of VGLUT1
positive particles is preserved in our FSC-triggered experiments.
VENUS
4.2.2 Evidence from Western blotting and immunofluorescence microscopy
positive particles is detected.FASS can enrich these fluorescently positive particles as analyzed by flow-cytometry.
After FASS in FSC-mode, which analyzes particles in the size range of 0.75 to 1.5 µm polystyrene beads, the sorted VGLUT1VENUS positive particles were not only enriched in VGLUT1VENUS but also in VIAAT (inhibitory synaptosomes) and PLP (myelin) as analyzed by Western blotting. FASS of in FM-mode isolated VGLUT1VENUS particles in a smaller size range and also enriched VGLUT1VENUS as assessed by Western blotting. In stark contrast to the isolation in FSC-Mode, FASS in FM-mode, depleted in VIAAT (inhibitory synaptosomes), VAChT (cholinergic synaptosomes), PLP (myelin), GLT1 (astrocytic membranes), IBA1 (microglial particles) and VGLUT2 (other excitatory synaptosomes). Furthermore, Western blot analysis of VGLUT1VENUS
Through this set of Western blot experiments I therefore established that VGLUT1
positive particles isolated by FASS in FM-mode, showed that they also contain proteins corresponding to all functional elements expected in VGLUT1 synaptosome, including: (i) several SV-proteins and SV-associated proteins (Synaptophysin, Synapsin, SV2B) (ii) proteins involved in endocytosis of VGLUT1 SVs (EndophilinA1) (iii) Components of the release machinery (SNAP23/25/47 and ComplexinII) (iv) mitochondrial proteins (VDAC1, TOM20) (v) components of the post-synaptic membrane (NL1, NR1, NR2A GluR2).
VENUS synaptosomes isolated by FASS in FM-mode allowed the specific enrichment
of VGLUT1VENUS
4.2.3 Evidence from electron and immunofluorescence microscopy
synaptosomal proteins, while depleting proteins of other synaptosomes and glial contaminants.
Beyond biochemical identification of protein markers we wished to assess the integrity and purity of synaptosomes isolated by FASS through photonic and electron microscopic methods. In a recent publication, Fein and coworkers, sorted synaptosomal particles in the size range of .75-1.5 µm beads in FSC-mode and processed them for EM by negative staining with uranyl acetate (Fein et al., 2008). The images shown do not reveal the ultrastructure of the isolated particles, but merely demonstrate the presence of round membrane bounded particles of varying sizes (no further quantification of the data was provided). An accepted view of the size of small CNS synaptic elements is of 0.5 to 2 µm as a whole with the presynapse being bigger than the post-synaptic specialization.
It may seem confounding that I detected synaptosomes in a size range below 0.75 µm in the calibration using light scattering of polystyrene beads in flow-cytometry. However, it is known that plastic beads and cells (synaptosomes) differ significantly in their refractive indices and therefore scatter light differently(Hoffman, 2005). Therefore, plastic beads only provide relative size references and can not easily be used to determine particle size exactly (Hoffman, 2005). Consequently the actual size of particles isolated by FASS may be larger than 0.75 µm. In agreement with this, my immunofluorescence and electron microscopy analyses of sorted VGLUT1VENUS synaptosomes demonstrated that most VGLUT1VENUS
Furthermore, we could show that 75 % of VGLUT1
synaptosomes are detected within a size range of 0.5 to 2 µm. These results indicate that the particles we isolated are in a size range that is expected for synaptosomes and that the calibration with polystyrene beads underestimated the size of synaptosomes.
VENUS particles isolated through FASS can be stained with antibodies against the postsynaptic marker PSD-95. As expected, VGLUT1VENUS and PSD-95 were not strictly colocalized, but rather facing each other. This indicated that at least 75 % percent of VGLUT1VENUS positive particles have a postsynaptic density attached and further validates that the isolated particles correspond to intact synaptosomes. It would theoretically be possible that some VGLUT1VENUS positive particles have postsynaptic densities attached that contain little or no PSD-95. Therefore, the fraction of VGLUT1VENUS
Additionally electron microscopy of VGLUT1
synaptosomes with a PSD attached might even be higher than 75 %.
VENUS FASS samples showed that they contain intact synaptosomes, which can be identified by their size, vesicular and mitochondrial content presynaptically and the presence of a post-synaptic density. A quantification of systematically taken EM images revealed that FASS enriches in particles with a synaptic profile, while depleting non-synaptic particles.
To summarize, the results obtained by microscopy convincingly demonstrate the enrichment in single, intact VGLUT1VENUS
4.2.4 Implications for the interpretation of earlier studies
synaptosomes of a size range compatible with the average synaptic sizes in the CNS.
The present results demonstrate the presence of single, intact VGLUT1VENUS