Figure 1. Super-resolution microscopy of fast-spiking, PV+ interneuron axons.

(a) Experimental approach. Biocytin-filled fast-spiking PV+ interneurons from mPFC were analyzed using both confocal imaging and structured illumination microscopy (SIM) imaging. See also Figure 1—figure supplements 1–3. (b) Maximum projection confocal image of a representative biocytin-filled PV+ cell from mPFC layer V (red). Scale bar, 50 µm. (c) Current clamp recording of evoked action potentials. Scale bars are 20 mV, 100 pA and 100 ms from top to bottom (right). (d) Full reconstruction of a mPFC layer V PV+ interneuron. Soma and dendrites in black, axon in brown. (e) Representative SIM z-stack projections of PV+ interneuron axonal segments (top), along with their corresponding FWHM diameter profiles (bottom). White arrowheads indicate measurement boundaries. From left to right: First branch order axon initial segment; second branch order unmyelinated axonal segment; third branch order myelinated axonal segment; sixth branch order unmyelinated axonal segment featuring multiple en-passant boutons (indicated by asterisks). Scale bar, 10 µm. (f) Distribution histogram of PV+ interneuron axon shaft diameters, fitted with a Gaussian curve. n = 140 axonal segments/8 cells. (g) Average axon shaft diameter decreases steadily over centrifugal branch order. n = 140 segments/8 cells. p<0.001, one-way ANOVA. (h) Distribution of axonal en passant bouton diameters of PV+ interneuron axons, fitted with a Gaussian curve. n = 250 boutons/8 cells. Abbreviations: FWHM, full-width half-maximum. I, input current. SIM, structured illumination microscopy. Vm, membrane voltage.

Figure 1—figure supplement 1. Experimental flowchart for axonal reconstructions.

Figure 1—figure supplement 2. Axonal diameter analysis.

Interbranch axonal diameter was sampled at high spatial frequency from branch point to branch point. This representative example based on Figure 1e, depicts a 3^rd order branch segment. (a) Axonal diameter was measured using the average intensity projection of a z-stack series of SIM images (red). A semi-automated, user-guided line was traced from the center of each axonal branch point to the center of the subsequent distal branch point (thin white line). Each segment was analyzed in the direction centrifugally oriented from the soma along the axon, as indicated by the white arrow. For every consecutive 40 nm of axon, a 50-pixel wide line (~2.0 µm) was drawn orthogonally to the tangent line of axonal orientation (white). Scale bar, 5 µm. (b) Along the orthogonals, pixel fluorescence intensities were extracted (black diamonds) and a Gaussian line was fitted (grey). Fits with r² <0.9 were excluded from further analysis. From the Gaussian fit at each consecutive axonal position, the full-width at half-maximum (FWHM) was calculated. In this representative example, the FWHM (axonal diameter)=0.238 µm and r² = 0.987. (c) Left axis: Axonal diameter measurements as a function of centrifugal distance along the axonal segment shown in (a). Right axis: Fit (r²) of the Gaussian function along the axonal segment. Black arrows indicate the location of the corresponding fit shown in (b). (d) Distribution over branch order of median Gaussian fit (r²) prior to exclusion of points with r² <0.9. Individual cells in grey, average in black. (e) Goodness of fit (r²) is similar between myelinated and unmyelinated segments. Individual cells in grey, average in black. Abbreviations: FWHM, full-with at half-maximum.

Figure 1—figure supplement 3. Locations of biocytin-filled and reconstructed PV+ and SOM+ cells.

(a) Maximum projection confocal image of a biocytin-filled mPFC SOM+ interneuron (red), with the distance to midline indicated. (b–e) Soma locations of Pvalb::cre,Ai14 cells (b), PV::UBE3A, PV::WT, PV::TSC1 cells (c), Sst::cre,Ai14 cells (d), and SOM::WT, SOM::TSC1 cells (e). Black bars indicate mean ± s.e.m.