Evaluating the inhibitory priority of Bcl-xL to Bad, tBid and Bax by using live-cell imaging assay.

Molecular regulatory network among the B cell leukemia-2 (Bcl-2) family proteins is a research hotspot on apoptosis. The inhibitory priority of anti-apoptotic Bcl-2 family proteins (such as Bcl-xL) to pro-apoptotic Bcl-2 family proteins (such as Bad, tBid and Bax) determines the outcome of their interactions. Based on over-expression model system, we here evaluate the inhibitory priority of Bcl-xL to Bad, tBid and Bax by using live-cell imaging assay on cell viability. Fluorescence images of living cells co-expressing CFP-Bcl-xL and YFP-Bad or YFP-tBid or YFP-Bax showed that Bcl-xL markedly inhibited Bad/tBid/Bax-mediated apoptosis, revealing that Bcl-xL inhibits the proapoptotic function of Bad, tBid and Bax. In the case of equimolar co-expression of Bad and CFP-Bcl-xL, the inhibition of Bcl-xL on tBid/Bax mediate-apoptosis was completely relieved. Moreover, co-expression of tBid-P2A-CFP-Bcl-xL significantly relieved the inhibition of Bcl-xL on the pro-apoptotic ability Bax, suggesting that Bcl-xL preferentially inhibits the pro-apoptotic ability of Bad over tBid, subsequently to Bax.

Spatial Distribution of Inhibitory Innervations of Excitatory Pyramidal Cells by Major Interneuron Subtypes in the Auditory Cortex.

Mental disorders, characterized by the National Institute of Mental Health as disruptions in neural circuitry, currently account for 13% of the global incidence of such disorders. An increasing number of studies suggest that imbalances between excitatory and inhibitory neurons in neural networks may be a crucial mechanism underlying mental disorders. However, the spatial distribution of inhibitory interneurons in the auditory cortex (ACx) and their relationship with excitatory pyramidal cells (PCs) remain elusive. In this study, we employed a combination of optogenetics, transgenic mice, and patch-clamp recording on brain slices to investigate the microcircuit characteristics of different interneurons (PV, SOM, and VIP) and the spatial pattern of inhibitory inhibition across layers 2/3 to 6 in the ACx. Our findings revealed that PV interneurons provide the strongest and most localized inhibition with no cross-layer innervation or layer specificity. Conversely, SOM and VIP interneurons weakly regulate PC activity over a broader range, exhibiting distinct spatial inhibitory preferences. Specifically, SOM inhibitions are preferentially found in deep infragranular layers, while VIP inhibitions predominantly occur in upper supragranular layers. PV inhibitions are evenly distributed across all layers. These results suggest that the input from inhibitory interneurons to PCs manifests in unique ways, ensuring that both strong and weak inhibitory inputs are evenly dispersed throughout the ACx, thereby maintaining a dynamic excitation-inhibition balance. Our findings contribute to understanding the spatial inhibitory characteristics of PCs and inhibitory interneurons in the ACx at the circuit level, which holds significant clinical implications for identifying and targeting abnormal circuits in auditory system diseases.