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. 2012;7(4):e35323.
doi: 10.1371/journal.pone.0035323. Epub 2012 Apr 23.

Synaptotagmin-2 is a reliable marker for parvalbumin positive inhibitory boutons in the mouse visual cortex

Jean-Pierre Sommeijer  1 , Christiaan N Levelt
Affiliations

Synaptotagmin-2 is a reliable marker for parvalbumin positive inhibitory boutons in the mouse visual cortex

Jean-Pierre Sommeijer et al. PLoS One. 2012.

Erratum in

  • PLoS One. 2012;7(7): doi/10.1371/annotation/1c5484e5-41c0-44dc-8422-2dbd3a002f3b

Abstract

Background: Inhibitory innervation by parvalbumin (PV) expressing interneurons has been implicated in the onset of the sensitive period of visual plasticity. Immunohistochemical analysis of the development and plasticity of these inhibitory inputs is difficult because PV expression is low in young animals and strongly influenced by neuronal activity. Moreover, the synaptic boutons that PV neurons form onto each other cannot be distinguished from the innervated cell bodies by immunostaining for this protein because it is present throughout the cells. These problems call for the availability of a synaptic, activity-independent marker for PV+ inhibitory boutons that is expressed before sensitive period onset. We investigated whether synaptotagmin-2 (Syt2) fulfills these properties in the visual cortex. Syt2 is a synaptic vesicle protein involved in fast Ca(2+) dependent neurotransmitter release. Its mRNA expression follows a pattern similar to that of PV throughout the brain and is present in 30-40% of hippocampal PV expressing basket cells. Up to now, no quantitative analyses of Syt2 expression in the visual cortex have been carried out.

Methodology/principal findings: We used immunohistochemistry to analyze colocalization of Syt2 with multiple interneuron markers including vesicular GABA transporter VGAT, calbindin, calretinin, somatostatin and PV in the primary visual cortex of mice during development and after dark-rearing.

Conclusions/significance: We show that in the adult visual cortex Syt2 is only found in inhibitory, VGAT positive boutons. Practically all Syt2 positive boutons also contain PV and vice versa. During development, Syt2 expression can be detected in synaptic boutons prior to PV and in contrast to PV expression, Syt2 is not down-regulated by dark-rearing. These properties of Syt2 make it an excellent marker for analyzing the development and plasticity of perisomatic inhibitory innervations onto both excitatory and inhibitory neurons in the visual cortex.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Syt2 and PV expression in the mouse brain.
(A–C) Localization of Syt2 and PV mRNA in the mouse brain according to the Allen Brain Atlas –. 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 figures 2–4 and 7&8 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.
Figure 2
Figure 2. Syt2 in L2-3 is expressed in PV positive boutons.
(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.
Figure 3
Figure 3. Syt2 in L5 is expressed in PV positive boutons.
(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.
Figure 4
Figure 4. Syt2 in L2-3 and L5 is not expressed in VGLUT1&2 positive boutons.
(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.
Figure 5
Figure 5. Quantification of colocalization of Syt2 and markers for specific subsets of inhibitory and excitatory neurons.
(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.
Figure 6
Figure 6. Scatter plot pixel analyses of Syt2 colocalization with cell type specific markers in various cortical areas.
(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.
Figure 7
Figure 7. Syt2 and PV expression at P18 and with dark-rearing.
(A–C) In L2-3 of the mouse visual cortex Syt2 is expressed in synaptic boutons at P18 when expression of PV is still hardly detectable in neurites or boutons. Arrowheads indicate Syt2 boutons without PV expression. The arrow points to an emerging PV punctum that also expresses Syt2. (D–F) In L2-3 of the visual cortex of dark reared mice from birth to P46 Syt2 expression is not reduced at P46 (D) while PV expression is reduced and not clearly localized in perisomatic boutons (compare to Fig. 2J–L) at the same synapses (E&F). The arrowheads indicate two boutons that both express PV and syt2, but he arrows point to two boutons that are not positive for PV. The asterisk marks a PV+ soma. A–C n = 2; D–F n = 3. Scale bar 10 µm.
Figure 8
Figure 8. Syt2 is expressed around eye-opening in VGAT positive boutons.
(A–C) Before eye-opening Syt2 colocalizes with VGAT+ boutons in L2-3 of the visual cortex. This colocalization continues after eye-opening, but well before onset of PV expression (D–I, and also Fig. 7B). A–C n = 3; D–F n = 2; G–I n = 3. Scale bar 10 µm.
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