Electron microscopy

Vibratome sections from the weaning-aged (PN23) and ontogeny study at ~Bregma −2.56 to −2.8 mm were taken for further analysis using the electron microscope (EM). These sections were immunostained for PV as described above, and then processed for EM (Farb et al., 1995; Aoki et al., 2000; Wable et al., 2014). Specifically, after terminating the HRP-DAB reaction, tissue was washed with 0.01M PBS and then post-fixed with 2% glutaraldehyde for 16 min at room temperature. Tissue was then washed in 0.01M PBS, washed again in 0.1M PB, transferred to ceramic multi-well dishes (Coors) and incubated for 1 hr in 1% osmium tetroxide (OsO₄). After rinsing with 0.1M PB, tissue was rapidly dehydrated in increasing concentrations of ethanol (30%, 50%, and 70%) before overnight incubation in 70% ethanol containing 1% uranyl acetate (UA) at 4°C while photoprotected by foil. Tissue was then rinsed in 70% ethanol and further dehydrated in 90% and 100% ethanol (2X), acetone Acetone ACS grade, Electron Microscopy Sciences (EMS); 3X), before immersion in 1:1 acetone: EPON (EMbed 812 embedding resin, DDSA, NMA, DMP30, EMS) for 1 hr at 50°C and 3 hrs at room temperature. After overnight incubation in 100% EPON, tissue was flat-embedded between Aclar sheets, weighted and cured at 60°C for ~36 hrs. BLA from flat-embedded tissue was then capsule-embedded and sectioned into 70 nm ultrathin sections with an ultramicrotome (MT-7, RMC Boeckeler). Sections were mounted on nickel grids (EMS cat# 15800) that had been coated in 0.3% Formvar (EMS) in ethylene dichloride under low-humidity conditions, and then counterstained with Reynold’s lead citrate.

All EM image acquisitions and quantifications were performed by persons blind to experimental condition. From each of six SAM and six control (CON) brains at age P23, cell bodies of 10 PV-immunoreactive inhibitory interneurons and 10 pyramidal neurons were sampled from the anterior-to-mid level of the BLA. Thus, from each animal, a total of 20 cell bodies were sampled. Cell bodies were imaged at 20,000X using the JEOL JEM1200-EX-II electron microscope and captured with the XR80 CCD camera and AMT software (Woburn, MA). To limit cell selection to the BLA subnucleus, distinguishing features of each BLA were mapped at 4X using an Olympus camera lucida. Images of different ultrathin sections were aligned after image acquisition to ensure that the same cell was never re-analyzed from another grid. To ensure full DAB immunoreactivity of PV cells and terminals, cell profiles were only image-captured close to the EPON-tissue interface carved by the vibratome blade, where immunolabeling could be expected to be optimal. DAB labeling was only considered positive if the soma or terminal was darker than the relatively electron-dense mitochondria in the imaged region.

Cell bodies in the BLA were categorized as one of three types. Pyramidal cells were identified as distinct from interneurons by their characteristically smooth nuclear envelope, lack of asymmetric axo-somatic synapses with thick post-synaptic densities (PSDs) and lack of PV-immunoreactivity. PV cells were identified by the presence of intense immunoreactivity of the perikaryal cytoplasm to the PV-antibody. The non-PV inhibitory interneurons were identified as such, based on the lack of PV-immunoreactivity, the presence of deep indentations along the nuclear envelope and presence of asymmetric axo-somatic synapses. Cell bodies of non-PV inhibitory interneurons were excluded from analysis for the current study.

Axon terminals forming axo-somatic synapses in the BLA were also categorized as one of three types: PV-immunoreactive and symmetric, PV-immunonegative and symmetric and PV-immunonegative and asymmetric, the last of which were considered to belong to excitatory neurons.

For each cell body profile, three measurements were made. The lengths of axon terminals’ plasma membrane forming direct contact with the cell body, i.e., free of astrocytic process intervention and less than 50 nm in distance between the axonal and cell body plasma membranes, were measured. We also measured lengths of the plasma membrane of the cell body. Both of these length measurements were made using digitally captured images using the Image J software (NIH, version 1.51). These values were used to obtain the average value of axon terminal lengths for that cell and the percent of plasma membrane of that cell body contacted by axo-somatic synapses of each type. The third measurement made for each cell body was the number of axon terminals forming axo-somatic synapses, per µm of cell body plasma membrane. Each neuron was considered an independent unit that could respond to alterations in levels of afferent activity from a variety of neurons within and outside of the BLA (i.e., N= cell). Thus, for statistical analyses, the average value of these measurements were assessed for each cell, and these ‘per cell’ values were pooled across six SAM brains. Similarly, neurons of each type of cell were pooled from six brains of the control group (CON).