Fig 2: Effects of NIR laser treatment after OGD toxicity on proteins involved in excitatory synaptic activity. Experiments were conducted as described in Fig. 1. Hippocampal slices were exposed to 30 min OGD and immediately after to 7.42 J/cm2 NIR laser. Twenty-four hours later, the expression of GluN1, PSD95, vGluT1, and EAAT2 was evaluated in total homogenate by Western blot analysis. Data are expressed as percentage of control. Bars represent the mean ± SEM of at least three experiments run in ottuplicate. *P < 0.05, **P < 0.01 vs. CRL, #P < 0.05, ##P < 0.01 vs. OGD (ANOVA + Tukey’s w test)

Fig 3: Differential Expression of Syt1 and Syt2 between Excitatory and Inhibitory Presynaptic Terminals in Cerebellum(A) Light-micrographs of cerebellar cortex, showing immunolabeling for synaptotagmin 1 (Syt1, left), synaptotagmin 2 (Syt2, center), and overlay (right) from a wild-type mouse; single confocal sections.(B) Similar to (A), but at higher magnification. Note that putative BC terminals surrounding PC somata are only immunoreactive for Syt2, but not for Syt1.(C) Colocalization of Syt1 and VGAT (left) and VGLUT1 (right). Note the absence of Syt1 immunoreactivity in inhibitory terminals surrounding PCs.(D) Similar colocalization analysis as shown in (C), but for Syt2. Note high Syt2 immunoreactivity in putative BC terminals (arrowheads).ML, molecular layer; PCL, Purkinje cell layer; GCL, granule cell layer.

Fig 4: Flow cytometric synaptosome analysis and gene expression of neurotrophins and neurotrophin receptors in naive and IAV-infected mice. (a to f) Synaptosomes were isolated from cortex (CTX) and hippocampal formation (HPF) of naive and IAV-infected mice and subjected to further flow cytometric analysis as shown in Fig. 7. (a and b) After size gating and removal of unspecific events via fluorescence-triggered detection (not shown), Synaptophysin+ (Syp+) events were selected and subsequently gated for Homer1+ or Gephyrin+, respectively. (c and d) Bar charts show the frequencies of Syp+ Homer1+ and Syp+ Gephyrin+ subpopulations from naive (white bars) and IAV-infected animals (black bars) at 14 and 21 dpi. (e and f) Intact synaptosomes from excitatory synapses consisting of a pre- and postsynaptic terminal (Syp+ Homer1+) were further examined for their expression of VGLUT1 and GluR1. (g to j) RNA was isolated from brains of perfused animals as described in the text. Relative gene expression levels of BDNF (Bdnf), NGF (Ngf), TrkB (Ntrk2), and p75NTR (Ngfr) were examined by RT-qPCR at different time points after IAV infection (10 to 21 dpi). Expression of target genes was normalized to the expression level of Hprt. Subsequently, relative expression was normalized to the means of naive animals. For all graphs, data are shown as means ± SEM, and groups were compared via Student's t test with Welch’s correction. Significant differences are indicated. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Fig 5: New tool to analyze synaptic proteins via flow cytometry. (a) Gene expression of VGLUT1 (Slc17a7) was analyzed in whole-brain homogenate of naive (white bars) and IAV-infected mice at 10, 14, and 21 dpi (black bars). Relative gene expression was examined by RT-qPCR as described in the text, and expression of the target gene was normalized to the expression level of Hprt. Subsequently, relative expression was normalized to the mean of naive animals. Groups were compared via Student's t test with Welch’s correction. (b) Synaptosomes were isolated from brain regions of perfused animals as described in the text, and the synaptosomal fraction (31) was subjected to electron microscopy. Red arrows indicate intact synapses formed by a pre- and postsynaptic compartment. Scale bar in the bottom left corner indicates 500 nm. (c) Graphical illustration of synaptosome selected in panel b. Presynaptic nerve endings still contain synaptic vesicles and mitochondria, whereas postsynaptic domains are characterized by their region of high postsynaptic density (PSD). (d and e) Protein content of VGLUT1 from synaptosomes of whole-brain homogenate was assessed via Western blotting. Bar charts show the relative optical density of the protein bands from naive (white bars) and infected (black bars) animals at 14 and 21 dpi upon normalization to ß-actin expression. Values were further normalized to the mean of naive animals. Groups were compared via one-way ANOVA with Holm-Sidak post hoc correction. (f to h) Isolated synaptosomes were subjected to flow cytometry, and the representative gating strategy is shown. (f) First, a gate with a size range from 300 to 1,000 nm was established in the FSC channel using silica beads. (g) Second, separation of biological particles from residues in the buffer was facilitated using the styryl dye FM4-64 that integrates into the lipid membranes of biological organelles. FSC-triggered detection then was replaced by fluorescence-triggered detection with FM4-64 in the BL3 channel with a fluorescence threshold set above the noise at 0.3 × 103 (not shown). (h) Lastly, events detected in the size range of 300 to 1,000 nm were further gated for their expression of VGLUT1. (i) Bar chart shows the frequency of VGLUT1+ events in the brains of naive (white bars) and IAV-infected animals (black bars) at 14 and 21 dpi. In all cases, data are shown as means ± SEM and significant differences are indicated. *, P < 0.05; ** P, < 0.01; *** P, < 0.001; **** P, < 0.0001.