Puncta labeling and imaging

Immunohistochemical methods were similar to those previously described (Hegarty et al., 2010, 2014). Solutions were prepared in either 0.1 m phosphate buffer at pH 7.4 (PB) or 0.1 m Tris-buffered saline at pH 7.6 (TS). Tissue sections were first rinsed in PB then incubated in 1% sodium borohydride in PB for 30 min to reduce background. After rinses in PB and TS, sections were incubated in 0.5% bovine serum albumin (BSA) in TS for 30 min and then placed in a primary antibody cocktail made in 0.1% BSA and 0.25% Triton X-100 in TS for 2 nights at 4°C. The primary antibody cocktail consisted of goat anti-glutamic acid decarboxylase 65/67 (GAD65/67; 1:100; catalog #sc-7513, Santa Cruz Biotechnology; RRID:AB_2107745), rabbit anti-parvalbumin (1:1000, catalog #NB120-11427, Novus Biologicals; RRID:AB_791498), and guinea pig anti-vesicular glutamate transporter 1 (VGluT1; 1:5000; catalog #AB5905, EMD Millipore; RRID:AB_2301751). After 40 h of primary antibody incubation, tissue sections were rinsed in TS and then incubated with a cocktail of fluorescently labeled secondary antibodies for 2 h, light protected, at room temperature. The secondary antibody cocktail consisted of Alexa Fluor-488 donkey anti-goat (1:800; catalog #A11055, Thermo Fisher Scientific; RRID:AB_2534102), Alexa Fluor-546 donkey anti-rabbit (1:800, catalog #A10040, Thermo Fisher Scientific; RRID:AB_2534016), and Alexa Fluor 647 donkey anti-guinea pig (1:800, catalog #706-605-148, Jackson ImmunoResearch; RRID:AB_2340476). Tissue sections were rinsed again in TS and then incubated in biotinylated WFA (1:50; catalog #B1355, Vector Laboratories; RRID:AB_2336874) for 2 h at room temperature. Following TS rinses, tissue sections were incubated for 3 h at room temperature in Alexa Fluor-405-conjugated streptavidin (6.25 µg/ml; catalog #{"type":"entrez-protein","attrs":{"text":"S32351","term_id":"1083459","term_text":"pir||S32351"}}S32351, Thermo Fisher Scientific). Finally, tissue sections were rinsed in TS followed by PB before being mounted with 0.05 m PB onto gelatin-coated slides to dry. Slides were coverslipped with Prolong Gold Antifade Mountant (Thermo Fisher Scientific) and light protected until imaging.

The anatomic landmarks used were those described above for PV and WFA labeling. To determine representative caudal and rostral sections of prelimbic cortex that were within +3.5 to +4.2 mm from bregma (Paxinos, 1998), two high-magnification images were taken at each level of the prelimbic cortex (2 images/level × 2 levels/animal = 4 images/animal). Images were captured on a Zeiss LSM 780 confocal microscope with a 63× 1.4 NA Plan-Apochromat objective (Carl Zeiss MicroImaging) using the single-pass, multitracking format at a 1024 × 1024 pixel resolution. Optical sectioning produced z-stacks bounded by the extent of fluorescent immunolabeling throughout the thickness of each section. Using Zen software (Carl Zeiss; RRID:SCR_013672), PV neurons in each confocal stack were identified and assessed for the presence of a nucleus and whether the entire neuron was within the boundaries of the field of view; only these PV neurons were included in the analysis. The optical slice through the nucleus at which the ellipsoidal minor axis length of each PV neuron reached its maximum was determined. A z-stack of that optical slice plus one optical slice above and one below was created, resulting in a 1.15 µm z-stack through the middle of each PV neuron; these subset z-stacks were used for puncta apposition analysis.

Image analysis of GABAergic and glutamatergic appositions onto PV-labeled neurons was performed using Imaris 8.0 software (BitPlane USA; RRID:SCR_007370) on an off-line workstation in the Advanced Light Microscopy Core at Oregon Health & Science University by a blinded observer. For each PV neuron, the manual setting of the Surfaces segmentation tool was used to trace the outline of the PV neuron in all three optical slices, and a surface was created. To limit our analyses to the area immediately surrounding each PV neuron, we used the Distance Transform function followed by the automated Surfaces segmentation tool to create another surface 1.5 µm away from the PV neuron surface that followed the unique contours of that PV neuron. The Mask Channel function was then used to examine only WFA, GAD65/67, and VGluT1 within this 1.5-µm-wide perimeter surrounding the PV neuron surface.

The presence of WFA labeling in close proximity to the PV neuron surface was assessed for each PV neuron. A PV neuron was considered to have a PNN if there was any WFA labeling around any part of the PV neuron surface as seen by the observer. GAD65/67 and VGluT1-labeled puncta were then assessed separately using the Spots segmentation tool. Within the Spots tool, the Different Spot Sizes (Region Growing) option was selected and initial settings included an estimated x–y diameter of 0.5 µm and an estimated z-plane diameter of 0.4 µm. Spots generated by Imaris from these initial settings were then thresholded using the Classify Spots, Quality Filter histogram to ensure that all labeled puncta were included and background labeling was filtered out. The spots were then thresholded using the Spot Region, Region Threshold histogram to ensure that the sizes of the Imaris-generated spots were good approximations of the size of the labeled puncta seen visually by the human observer. Using the Find Spots Close to Surface Imaris XTension, we then isolated those spots that were within 0.5 µm of the PV neuron surface. All segmented spots close to the PV neuron surface had to have a z-diameter of at least 0.4 µm to be considered puncta (Hegarty et al., 2010, 2014).