Syt2 in L2-3 and L5 is not expressed in VGLUT1&2 positive boutons.

<p>(A–F) Syt2 puncta (arrowheads) in L2-3 and L5 are not VGLUT1 positive. Arrows indicate VGLUT1 boutons. VGLUT1 and Syt2 show an alternating perisomatic localization (F). Similarly VGLUT2 boutons, indicated with arrows do not show expression of Syt2. Syt2 puncta indicated with arrowheads (G–L). A–L n = 3. Scale bars 10 µm.</p

Syt2 in L2-3 is expressed in PV positive boutons.

<p>(A–C, arrowhead) Syt2 almost always colocalizes with VGAT in L2-3 of the visual cortex. The arrow indicates a VGAT positive bouton without Syt2 expression. (D–I) Syt2 puncta (arrowhead) are not calretinin (CR, arrow) or somatostatin (SOM, arrow) positive. (J–L) Syt2 shows strong co-expression with PV (arrowheads). A–C & G–I n = 3; D–F & J–L n = 4. Scale bars 10 µm.</p

Percentages of colocalization of Syt2 with different markers.

<p>Values represent e.g. the mean percentages (± SEM) of Syt2 puncta that also expressed VGAT.</p

Syt2 in L5 is expressed in PV positive boutons.

<p>(A–C, arrowheads) Syt2 almost always colocalizes with inhibitory VGAT positive puncta in L5 of the visual cortex. The arrow indicates a VGAT positive punctum without Syt2 expression. (D–L) These inhibitory Syt2 puncta (arrowheads) are not calbindin positive (D–F), CR positive (G–I) or SOM positive (J–L) (arrows). (M–O) As in L2-3, Syt2 shows a high colocalization with PV positive puncta in L5 of the visual cortex (arrowheads). Note the PV+ neurite (arrow) which is without expression of Syt2 (M–O). A–C & J–L n = 3; D–I & M–O n = 4. Scale bars 10 µm.</p

Scatter plot pixel analyses of Syt2 colocalization with cell type specific markers in various cortical areas.

<p>(A) Example images of neurons with perisomatic punctate innervation from different areas of the cortex. Adjacent are scatter plots for the same images showing distribution of fluorescence intensities for Syt2 (vertical green channel) and PV (horizontal red channel). As a negative control for colocalization (B) shows examples of Syt2 positive innervations with VGLUT2 or CR. Adjacent are scatter plots showing distribution of fluorescence intensities for Syt2 (vertical green channel) and VGLUT2 or CR (horizontal red channel). (C) The average Manders' coefficients of all the scatter plots. Coefficient values in different cortical areas are similar to V1 for colocalization of Syt2 and PV, and differ strongly from non-colocalization values for Syt2 with VGLUT2 or CR (n = 8; n = 7&8). M1, primary motor cortex (n = 6&9); S1, primary somatosensory cortex (n = 7&10); S1BF, barrel field cortex (n = 11&11); Au1, auditory cortex (n = 4&9). Data in C are given in mean coefficients ± SEM. Scale bar 10 µm.</p

Syt2 and PV expression in the mouse brain.

<p>(A–C) Localization of Syt2 and PV mRNA in the mouse brain according to the Allen Brain Atlas <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035323#pone.0035323-Allen1" target="_blank">[26]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035323#pone.0035323-Lein1" target="_blank">[27]</a>. Note the resemblance between the two patterns. Syt2 mRNA is detectable in the Caudate Putamen (CPu), reticular thalamic nucleus (Rt), zona incerta (ZI) and ventromedial hypothalamic nucleus (VMH). Expression is also seen in the hippocampal areas and the subiculum, as well as in the superior colliculus (SC) and the molecular layer of the cerebellum (ML). A sparse but layered signal is seen in the visual cortex (V1) (C). (D) Syt2 protein expression is visualized in a coronal section of the mouse brain using the i735 antibody. The white box highlights the part of the visual cortex from which data for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035323#pone-0035323-g002" target="_blank">figures 2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035323#pone-0035323-g004" target="_blank">4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035323#pone-0035323-g007" target="_blank">7</a>&<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035323#pone-0035323-g008" target="_blank">8</a> are obtained. (E) Syt2 is expressed in all layers of the visual cortex, except for layer 1. It is localized in a perisomatic fashion around many cells in L2-3 – L6. Expression is strongest in the lower part of L5 where larger pyramidal neurons reside. Scale bar (C) 180 µm, (E) 50 µm.</p

Quantification of colocalization of Syt2 and markers for specific subsets of inhibitory and excitatory neurons.

<p>(A) Percentage of colocalization of VGAT in Syt2+ puncta in L2-3 and L5. (B) Percentage of calbindin colocalization in Syt2+ puncta (CB/Syt2), and Syt2 in calbindin+ puncta (Syt2/CB) in L5. (C) Percentage of colocalization of calretinin in Syt2+ puncta (CR/Syt2) and Syt2 in calretinin+ puncta (Syt2/CR). (D) Percentages colocalization of somatostatin in Syt2+ puncta (SOM/Syt2) and Syt2 in somatostatin+ puncta (Syt2/SOM). (E) Percentages colocalization of PV in Syt2+ puncta (PV/Syt2) and Syt2 in PV+ puncta (Syt2/PV). (F) Percentages colocalization of VGLUT1 or VGLUT2 in Syt2+ puncta (VGLUT1/Syt2 and VGLUT2/Syt2) and Syt2 in VGLUT1+ or VGLUT2+ puncta (Syt2/VGLUT1 and Syt2/VGLUT2). Data are given in mean percentage ± SEM.</p

Mosaic transgene expression in various parts of the adult forebrain.

<p>Epi-fluorescence microscopy images showing expression of EGFP-F in TLG 498⁺ Cre 3487⁺ transgenic mice at 2 months of age in (A) pyramidal cells in layers II/III and V/VI of visual cortex, C) pyramidal and granule cells of hippocampus, (E) granule cells of the olfactory bulb, (G) pyramidal cells of amygdala and (I) piriform cells in superior colliculus (arrows). There is no expression in the cerebellum (K). Scale bar −200 µm. A very similar expression pattern was observed in 2½ month old TLG 1157⁺ Cre 3487⁺ transgenic mice as shown in the juxtaposed figures (same scale): (B) pyramidal cells in visual cortex, (D) granule cells of hippocampus, (F) granule cells of the olfactory bulb, (H) pyramidal cells of amygdala, and (J) piriform cells in superior colliculus. Again, no expression was observed in cerebellum (L).</p

Generation of Cre-dependent mosaic transgenic mice.

<p>(A) Transgenic mice based on Cre/Lox recombination were generated by crossing a Cre-transgenic line under a CaMKIIα promoter with a Thy1 promoter-driven EGFP-F transgenic line containing a transcriptional STOP cassette. Bright field microscopy images showing mosaic expression of the β-galactosidase expressing cells in layers II/III and V/VI of the neocortex of R26R mice when crossed with different CaMKIIα promoter-driven Cre-transgenic lines – (B) Cre 3487 and (C) Cre 3510. Epi-fluorescence microscopy images of Cre-dependent EGFP-F expression in TLG 498⁺ Cre 3487⁺ transgenic mice. EGFP-F expression starts at 6 weeks and increases with age. The number of EGFP-F⁺ cells increases from (D) 2 months to (E) 4 months of age. Scale bars: B, D-100 µm; C-200 µm.</p

Colocalization of EGFP-F and β-galactosidase in TLG 498⁺ R26R⁺ Cre 3487⁺ mice.

<p>Two month old TLG 498⁺ R26R⁺ Cre 3487⁺ transgenic mice were analyzed for colocalization of β-galactosidase in EGFP-F⁺ cells of the visual cortex. A total of 8 sections of 100 µm thickness were used from 3 mice and 201 cells were counted. Numbers between brackets are absolute numbers of cells counted. Between 80–90% of neurons expressing β-galactosidase also express EGFP-F and vice versa.</p