Fig 2: Definition of the borders of the CA2 region. (A) GAD67 fluorescent staining (scale bar 1 mm). GAD-positive neurons in the CA1, CA2, and CA3 regions are shown at higher magnification in the bottom panels (scale bars 200 µm). SO, Stratum Oriens; SP, stratum pyramidale; SR, stratum Radiatum; SLM, Stratum Lacunosum Moleculare; SL, Stratum Lucidum. (B) Immunoperoxidase staining of parvalbumin (PV)-positive interneurons (scale bar 1 mm). (C) Immunofluorescence staining for a actinin 2 protein and purkinje cell protein 4 (PCP4) determined the borders of the CA2 region. Scale bars, 1 mm. (D) The borders of CA2 (white dotted line) were defined by superimposition of the PCP4 labeling and GAD67 and immunoperoxidase stainings.

Fig 3: Distribution of somatostatin-immunopositive interneurons in CA2. (A) Example of immunoperoxidase staining for somatostatin. Superimposition of this staining with the labeling of PCP4 (bottom right panel) defined the borders of the CA2 region (white dotted lines). White arrows indicate some immunopositive somata (top left panel). SOM-immunopositive somata in SP and SO of the CA2 region are shown at high magnification in the bottom left panel. Scale bars 1 mm (top right panel), 200 and 50 µm (top and bottom left panels respectively). SO, Stratum Oriens; SP, stratum pyramidale; SR, stratum Radiatum; SLM, Stratum Lacunosum Moleculare; SL, Stratum Lucidum. (B) Distribution of SOM-immunopositive somata in CA1, CA2, and CA3 (superimposition of five 50 µm-thick sections). (C) Distribution of SOM-positive neurons in all layers of the three CA regions. Number of neurons was expressed as percentage of GAD-positive neurons (5 sections- n = 6). SOM-positive somata were mainly located in SO, SP, and SL of the three regions, with a few cells in SR and very few in SLM. There was fewer SOM-positive cell bodies in SO and SP of the CA1 region than of the CA2 and CA3 regions (MWU P < 0.05). SR of the CA3 region also contained fewer SOM-immunopositive somata than in CA1 (MWU P < 0.05). *P = 0.01 to 0.05.

Fig 4: Visualization of CCK inhibitory neurons in BLA. (A) Expression of eGFP in BLA CCK inhibitory neurons by using CCK-IRES-Cre mice and AAV-Dlx-DIO-eGFP vector. (B) Most eGFP positive neurons (green) are GAD (red) positive (97%, 28/29) and (C) CamK2 (red) negative (97%, 70/72) in BLA. Scale bar=100 µm (left), 20 µm (right, magnified image). (D) ROIs are outlined in the boundary of soma of each GFP+ cells to calculate soma size. Scale bar=100 µm. (E) Individual soma sizes are shown. 176 µm2 (the size of cell with 15 µm in diameter) is used as criteria (red dashed line) for classification of type L CCK and type S CCK neurons. (F) Type L CCK neurons (size > 176 µm2) comprise 36% of total CCK neurons (106 cells), and remained portion (64%) are type S CCK neurons (size <176 µm2). (G) Soma size for each group is presented as median with 25th/75th percentiles (box) and 10th to 90th percentiles (whiskers).

Fig 5: CTA learning does not affect intrinsic excitability but reduces excitatory synaptic drive onto EXC neurons in L2/3 aGC.(A) Images of example recorded neurons from a control animal (top) and CTA animal (bottom). Left: biocytin (green), middle: GAD-67 (magenta), right: Merge showing lack of co-localization. Scale bar = 25 µm. (B) Steady-state firing patterns of neurons from a control animal (top) and CTA animal (bottom). (C) FI curves from control (N = 15 rats, 3–4 cells/animal) and CTA animals (N = 14 rats, 3–4 cells/animal). Average FI slope and input resistance did not differ between groups (FI slope: Control 0.07 ± 0.01 vs. CTA 0.09 ± 0.02, p=0.17; Input Resistance: Control 136.47 ± 13.62 MO vs. CTA 148.97 ± 24.59 MO, p=0.38). (D) Sample traces of sEPSCs (holding = -50 mV) onto a control neuron and CTA neuron. Average EXC charge was larger in the control group (Control, [N = 8 rats, 3 cells/animal] 16.09 ± 0.90 pC vs. CTA [N = 7 rats, 3–4 cells/animal] 13.32 ± 1.14 pC, p<0.01). (E) Cumulative probability plots showing a decrease in sEPSC amplitude (left), but not sEPSC instantaneous frequency (right) in the CTA group (sEPSC amplitude [n = 24 cells/group, a = 0.0004]: Control vs. CTA, p<10-11; sEPSC frequency [n = 24 cells/group, a = 0.0004]: Control vs. CTA, p=0.02). Average sEPSC amplitude and frequency (insets) did not differ between groups (sEPSC amplitude: Control, [N = 8 rats, 3 cells/animal] 20.23 ± 3.12 pA vs. CTA [N = 7 rats, 3–4 cells/animal] 17.94 ± 2.96 pA, p=0.32; sEPSC frequency: Control, [N = 8 rats, 3 cells/animal] 8.14 ± 5.17 Hz vs. CTA [N = 7 rats, 3–4 cells/animal] 9.55 ± 5.90 Hz, p=0.75). * indicates p=0.05. Error bars ± 95% CI. The source data reported in the figure are in the Figure 2—source data 1.Figure 2—source data 1.This file contains source data and statistics presented in each panel of Figure 2, Figure 2—figure supplement 1.Excel worksheet tabs are labeled to indicate the specific figure. Each data table is labeled to indicate the specific figure panel. The statistics worksheet indicates specific tests and values applied to each data set in the figure and supplements.