Fig. 5: The IBIST-based Ca²⁺ recording for cells defined by multiple projections.

a Scheme illustrating AAV injections to define cells by three features: dual retrograde tracings and pyramidal-cell-specific (CaMKIIα promoter) Ca²⁺ recording. b Schematic showing the behavioral design of water CPP and shock sessions. c Examples of fluorescence images in vCA1 (top, injected with AAV-TRE-GCaMP6s, shown in green) and downstream regions (bottom). AAVretro-tTAN (shown in magenta) and AAVretro-tTAC were injected into two downstream regions, co-injected into one region, or omitted. All the AAVs were co-injected with blue beads (shown in magenta). The arrows indicate optical fiber tracks. mPFC: medial prefrontal cortex. NAc: nucleus accumbens. Amy: amygdala. Scale bars: 500 µm. d–g, Examples of heatmaps and averaged traces of Ca²⁺ signals upon water reward (d, blue), sucrose reward (e, yellow), water omission (f, black) and foot shock (g, red). h Summary of Ca²⁺ responses in vCA1 depending on targeted areas. The significance for Ca²⁺ response in each group is determined by one sample t-test (H0: μ = 0), *P < 0.05, **P < 0.01, ***P < 0.001 . i, Polar summary plot of Ca²⁺ responses to foot shocks and water and sucrose rewards normalized by the max response in each type of emotional stimuli. Data summary: mean ± SEM, details in Supplementary Table 2. Statistical analysis: two sided, details in Supplementary Table 3. Source data are provided as a Source Data file.