Axo imager z2

Neurons were passively filled with biocytin in the whole-cell configuration. Following recordings, the pipette was carefully retracted, and the acute slice was placed in a petri dish between filter papers. Slices were fixed overnight with 4% PFA in PBS. Biocytin was revealed by treating the slices with Triton (1%) and incubating overnight in an Alexa-633 conjugated streptavidin (1:200, ThermoFisher Scientific). The following day, slices were mounted on microscope slides with ProLong Gold (ThermoFisher Scientific). Images were acquired on a Zeiss confocal system (Axo Imager.Z2). Anatomical tracings were performed in Neurolucida 360 (2.70.1, MBF Bioscience) on a personal computer.
For anatomical classification, the axonal length in the dendritic layers (strata oriens and radiatum) and in the somatic layer (stratum pyramidale) were quantified in Neurolucida. For each cell, axonal length was measured using Neurolucida 360. The axonal length in the somatic or dendritic layers were then normalized to the total axonal length for each cell. Using this dataset, K-means clustering analysis in Python was used to cluster interneurons in two groups.

For localization of K_V1.1 in PV-IN, 20 μm thick hippocampal slices from Pv-Ai9 animals were prepared on a cryostat (CM3050 S, Leica). Slices were treated with a K_v1.1 recombinant rabbit monoclonal antibody (SN66–06, ThermoFisher Scientific) overnight and with an Alexa-488 conjugated secondary antibody for two hours on the following day. Images were acquired on a Zeiss confocal system (Axo Imager.Z2). Pv-Ai9-expressing interneurons were considered positive for K_v1.1 if the Alexa-488 fluorescence intensity at the soma was two standard deviations above the surrounding background.