Fig. 5

ACC projection to the BLA is monosynaptic and glutamatergic. a Anterograde terminal tracing strategy based on expression of eGFP-Syb2. Confocal image shows mCherry-expressing cells selectively observed in the ACC. Scale bar, 500 µm. b Representative confocal images showing eGFP-Syb2 expression pattern in the amygdala sub-nuclei. Strong eGFP-Syb2 expression was observed in the BLA. Scale bar, 200 µm. c Quantitative analysis of the eGFP-Syb2 signals in the amygdala sub-nuclei (across three different mice). Fluorescence intensity was normalized to mean intensity value (0–255 scale) measured in the LA area. d Retrograde tracing strategy using CAV2-Cre-dependent expression of DIO-eYFP. e Representative confocal image showing eYFP-expressing cells in the ACC. Scale bar, 500 µm. f Quantitative analysis of the retrogradely labeled neurons (eYFP+) in the ipsilateral and contralateral ACC. g Representative confocal images showing VGLUT1 or VGAT immunoreactive signals with eGFP-Syb2 signals. Scale bar, 10 µm. h Bar graph showing co-localized puncta in the BLA. i Illustration of ex vivo patch-clamp recording experiments for monitoring light-evoked PSPs in ACC input-identified BLA neurons. j Representative epi-fluorescence image indicating the patch-clamp recording site. Bottom left image, magnification of the black square area. tdTomato-positive (tdT+) cells were selectively targeted, as indicated by red arrow. Scale bar, 500 µm. k Proportion of neurons displaying PSP responses to light stimulation. Total 17 tdT+BLA neurons were successful for whole-cell patch-clamp recording from 10 slices of 5 mice. 12 of 17 cells were PSP-positive (PSP+). l Representative recording traces indicating light-evoked PSPs in the ACSF (black), and in the presence of bicuculline (+bic; blue), bicuculline+4-AP+TTX (+4AP/TTX; green), or bicuculline+4-AP+TTX+NBQX+APV (+NBQX/APV; red). m PSP latency of the PSP+/tdT+BLA neurons recorded (n = 12 cells). n Summary of amplitudes of light-evoked PSPs measured from each drug treatment condition (n = 6 cells from 6 slices of 3 mice). Repeated measures one-way ANOVA (F_{(1.167, 5.837)} = 6.900, P = 0.0376) with Dunnett’s post hoc test (ACSF vs.+NBQX/APV; P = 0.0106). Data are expressed as mean ± s.e.m. *P < 0.05. o Anterograde tracing strategy based on transneuronal delivery of AAV1-hSyn-Cre. p Representative confocal images showing the pattern of tdT+expression in the ACC (left, scale bar: 500 µm) and the amygdala (right, scale bar: 200 µm). q Quantitative analysis of the tdT+neurons in the BLA and CeA. BMA, basomedial nucleus; LA, lateral nucleus; CeA (CeM, CeL, and CeC), central nucleus (medial, lateral and capsular subdivisions) of the amygdala¹⁵