bAP-Ca2+ transient enhancement depends on neuronal output.(a) Raster plot of a 10 min in vivo extracellular recording from a freely moving p19 rat in layer 2 of the MEC. Grey bars correspond to single spikes. AP doublets with frequencies >100 Hz are indicated in red. (b) Diagram of experimental timelines. Grey and black vertical bars correspond to test stimulus doublets (short bars) and the ten 5 AP bursts administered after baseline (long bars). Dark green vertical bars depict doublet test stimuli delivered every 60 s. A 6 min baseline measurement was followed by a stimulus-free interval of 5 min. We then again measured the bAP-Ca2+ transients evoked by the doublet test stimulus. Light green bars correspond to three doublet test stimuli delivered at an interval of 120s and followed by a 10 min stimulus-free interval. After that, six doublets were measured at a 60 s interval. Shorter pink horizontal bars correspond to singlets. For baseline measurements, three singlet test stimuli were delivered at an interval of 120 s and followed by a 10 min stimulus-free interval. After that, six singlets were measured at a 60 s interval. Interval used for normalized post/pre ratios of bAP-Ca2+ transient enhancement is shaded in grey. (c1) Z-projection of the imaged dendritic segment (scale bar corresponds to 1 μm). (c2) Singlet evoked averaged bAP-Ca2+ transients from two neighbouring spines (top and middle). Bottom trace refers to representative underlying APs. (d1) Plot of normalized bAP-Ca2+ transient enhancement 15 to 20 min after bAP stimulation onset in spines with baseline ∆G/R <0.041. In the singlet experiments, the doublet response was measured at 20 min to permit grouping for comparison with the other doublet responses. bAP-Ca2+ transient enhancement in the control group (black, n = 53/31 spines/cells) is significantly larger than in the spine group where singlets were applied (-16.5%, n = 16/11 spines/cells, magenta, p < 0.001, Kruskal Wallis Test with Dunn's posthoc comparison). Reduced enhancement upon application of doublets when 5 AP bursts were omitted (27 ± 8%, n = 10/6 spines/cells, dark green) and doublet number further reduced (14 ± 5%, n = 16/12 spines cells, light green). Both conditions were not significantly different from controls (n.s., Kruskal Wallis Test with Dunn's posthoc comparison). (d2) Cumulative distribution plot of normalized bAP-Ca2+ transient enhancement. Dataset corresponds to d1. Data are expressed as mean SEM *** P < 0.001.