Fig. 4: Properties of IC-BLA and IC-CeM synapses, and of IC-BLA and IC-CeM neurons.

a Experimental strategy of expressing ChR2-eYFP in the insular neurons to confirm the monosynaptic connection of the insular neurons to basolateral amygdala (BLA, IC-BLA) and medial subdivision of central amygdala (CeM, IC-CeM) and record the dynamic properties of insular synapses on BLA and CeM neurons upon the optogenetic activation of insular terminals. b Representative traces of synaptic responses upon the application of ACSF, +TTX + 4AP and +AP5 + NBQX in order to prove the monosynaptic connection of the insular neurons to the BLA and CeM neurons. c Quantification of optogenetic-induced excitatory/inhibitory postsynaptic currents (oEPSC/oIPSC) of BLA and CeM neurons upon optogenetic activations of insular axonal terminals during the application of ACSF, +TTX + 4AP and +AP5 + NBQX (Two-way ANOVA: drug effect for oEPSC: F_(2,40) = 44.08, p < 0.0001 from BLA n = 10 cells, CeM n = 12 cells; drug effect for oIPSC: F_(1,22) = 39.34, p < 0.0001 for BLA n = 12 cells, CeM n = 12 cells). d Latency to the peak of oEPSC or oIPSC (Two-tailed paired t-test, t = 9.111 ***p < 0.0001 for BLA n = 10 cells, t = 5.467, ***p = 0.0003 for CeM n = 11 cells). e Representative traces and summary data of paired pulse ratio at -70 mV (excitatory PPR) and 0 mV (inhibitory PPR) at the IC-BLA and IC-CeM synapses. (oEPSC: BLA n = 11 cells, CeM n = 12 cells, oIPSC: BLA n = 11 cells, CeM n = 11 cells). f, g Representative traces f and summary data g of excitatory or inhibitory responses of BLA and CeM neurons upon the 10 train stimulations of ChR2 in insular terminals (Repeated measures of ANOVA: Interaction effect for EPSC: F_(9,153) = 2.563, **p < 0.01, Interaction effect for IPSC: F_{(9, 180)} = 1.843, p = 0.0634). h. Experimental plan for CTB labeling and whole-cell patch recording of IC-BLA and IC-CeM. i. Representative images of CTB injection sites in BLA and CeM. j. Representative images of biocytin-filled neurons labelled by CTB. k, l Analysis of intrinsic properties from IC-BLA and IC-CeM neurons depending on their anterior-posterior k and layer l position. Measures were obtained from the membrane seal test [1], the IV curve [2], and the Ramp test [3]. See Supplementary Fig. 4a for example traces. k Membrane and input resistances were higher for pIC-BLA compared to aIC-BLA neurons (Two-tailed t-test, t = 2.415, *p = 0.025, and t = 2.154, *p = 0.042 respectively). The rheobase was higher in aIC-BLA neurons (Two-tailed t-test, t = 3.164, **p = 0.005). For aIC-BLA n = 15 cells, pIC-BLA n = 9 cells, aIC-CeM n = 5 cells and pIC-CeM n = 12 cells. l Membrane capacitance is higher for L5 than L2/3 IC-BLA neurons (Two-tailed t-test, t = 2.219, *p = 0.037). For IC-BLA L2/3 and L5 n = 12 cells, IC-CeM L2/3 n = 6 cells and IC-CeM L5 n = 11 cells. m Representative trace of output firing in response to input current steps, analyzed in the following panel. n aIC-CeM neurons are more excitable than pIC-CeM neurons (Two-way ANOVA, F_(1,15) = 5.958, *p = 0.023). All results are represented as mean ± SEM.