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. 2020 Oct 12;9:e59699. doi: 10.7554/eLife.59699

Figure 4. Adaptation of the workflow for rapid and specific isolation and metabolite profiling of synaptic vesicles (SVs) directly from mouse brain tissue.

(A) Schematic of the workflow used to isolate SVs from mouse brain tissue. (B) Immunofluorescence images of coronal sections from wild-type mouse brains transduced with SV-tag in the indicated brain regions. Neurons are labeled with DAPI nuclear stain (blue) and SV-tag (red). Scale bar: 1 mm. (C) Immunoblot analysis of indicated protein markers present in brain lysates, control immunoprecipitates from uninfected brains, and HA immunoprecipitates from hippocampi and striatum that were infected with SV-tag. (D) LC/MS profile of SVs isolated from wild-type mice brains infected with SV-tag in hippocampus compared to a control IP from uninfected brains. (mean ± SEM, n = 4). Color code and legend is the same as in Figure 2C. (E) LC/MS profile of SVs isolated from wild-type mice brains infected with SV-tag in striatum compared to uninfected brains. (mean ± SEM, n = 4) (F) Construct design for expression of SV-tag in neurons in a Cre-dependent manner. (G) LC/MS profile of SVs isolated from Slc32a1IRES-Cre/wt (VGATIRES-Cre) mice brains infected with SV-tag in striatum compared to uninfected brains. (mean ± SEM, n = 4) (H) Coronal sections from a Slc6a3IRES-Cre/wt mouse transduced with Cre-dependent SV-tag in dopaminergic neurons of the midbrain. Dopamine neurons are immunolabelled for tyrosine hydroxylase (TH, magenta), DAPI nuclear stain (blue) and SV-tag (red). (I) Targeted LC/MS profiling of dopamine in SVs isolated from Slc6a3IRES-Cre/wt mice transduced with Cre-dependent SV-tag in dopaminergic neurons of the midbrain. Indicated mice were subjected to saline injection or tetrabenazine injection intraperitoneally 2 hr prior to harvesting of SVs.

Figure 4—source data 1. Absolute dopamine concentrations measured for Figure 4I.

Figure 4.

Figure 4—figure supplement 1. Characterization of synaptic vesicles isolated directly from mouse brain tissue.

Figure 4—figure supplement 1.

(A) Immunofluorescence images of transverse sections containing hippocampal CA3 transduced with SV-tag. A cut was made between CA2 and CA1 (dashed white line) to prevent recurrent excitation of these synapses. (B) Representative traces of evoked current responses in hippocampus from electrical stimulation. Vertical blue bars indicate stimulation that occurred with an interstimulus interval of 50 ms of Schaffer collaterals with (red) and without (black) expression of SV-tag. Vertical bar indicates 100 pA. Non-significant p-value = n.s. (C) Summary of paired-pulse ratios of electrically evoked synaptic responses from CA3 to CA1 synapses that were uninfected or expressing SV-tag. (D) Immmunoblot analysis of indicated proteins for synaptic vesicles (SVs) isolated from the whole-cell lysis method of brains compared to vesicles isolated with the synaptosome method. (E) Immunoblot analysis of SVs isolated from mouse brains transduced with SV-tag in the cortex. (F) Electron microscope image of SVs generated with the workflow. Values denote diameter of indicated particles. Scale bar: 100 nm (G) Coronal sections from Slc32a1IRES-Cre/wt (VGAT IRES-Cre/wt) and Adora2aCre/wt mice transduced with a Cre-dependent SV-tag in the striatum. (H) LC/MS profile of SVs isolated from Adora2aCre/wt mice brains infected with SV-tag in striatum compared with uninfected brains. (mean ± SEM, n = 4) (I) Representative traces of amperometry demonstrating the effects of DMSO (black) and tetrabenazine (black) on evoked dopamine. Blue indicates electrical stimulation. (J) Amperometry for dopamine in coronal slices prepared from mice administered intraperitoneally with tetrabenazine (30 mg/kg) or vehicle control 2 hr before slicing.