Figure 4. Ex vivo calibration of GCaMP6s and GCaMP6f signals in soma and apical tuft dendrites.

(A) Experimental schemata. Layer 5 pyramidal neurons expressing GCaMP6s or GCaMP6f were recorded using whole-cell patch clamp in acute cortical slices of the primary visual cortex. An example neuron is shown (scale bar = 20 μm). The soma and distal apical dendrite (nexus) were imaged during neuronal spiking, driven either by suprathreshold somatic current injection (somatic stimulation) or layer 1 (L1) stimulation (dendritic stimulation), respectively. Somatic and dendritic stimulation consisted of a train of 10 pulses delivered at 5–200 Hz. (B) Examples of action potentials driven by somatic and dendritic stimulation. Action potentials triggered by dendritic stimulation were additionally accompanied by an enhanced afterdepolarization reminiscent of a dendritic spike, which had a faster rise time than subthreshold EPSPs and were absent from somatically-evoked action potentials. (C, D) Example calcium transients imaged in the soma and apical dendrite during somatic and dendritic stimulation in a L5 neuron expressing GCaMP6s (C) and GCaMP6f (D). Concurrent somatic electrophysiological recordings are shown below each transient. Negative deflections in electrophysiological traces are stimulation artefacts that have been cut for clarity. (E, F) Average peak amplitude of calcium transients recorded in the soma (red) and dendrites (blue) during somatic and dendritic stimulation for GCaMP6s (E) and GCaMP6f (F). Null distribution of peak amplitudes from sham stimulation trials are shown (dashed horizontal lines). (G, H) Proportion of compartment-specific events detected either in the soma or dendrite during somatic or dendritic stimulation for GCaMP6s (G) and GCaMP6f (H). Shaded areas represent S.E.M. Significance was assessed with two way (E, F) and one way (G, H) repeated measures ANOVA with post-hoc Sidak tests. Asterisks (***) in (E,F) denote factor (soma vs dendrite) significance of p<0.0001; n.s. denotes non-significance; significance is not shown for other comparisons but is listed below. For somatic stimulation in (E): soma vs. dendrite: p<0.0001 for all frequencies; soma vs. null: p<0.0001 for all frequencies; dendrite vs null; p<0.0001 for frequencies > 50 Hz, p>0.44 for frequencies < 50 Hz. For dendritic stimulation in (E): soma vs. dendrite: p>0.11 for all frequencies; soma vs null: p<0.0001 for all frequencies; dendrite vs null: p<0.0001 for all frequencies. For somatic stimulation in (F): soma vs. dendrite: p>0.49 for>25 Hz, p=0.074 for 10 Hz, p=0.054 for 5 Hz; soma vs. null: p<0.0001 at>100 Hz, p=0.09 at 50 Hz, p>0.7 at<50 Hz; dendrite vs. null: p<0.0001 at>100 Hz; p>0.52 at<50 Hz. For dendritic stimulation in (F): soma vs. dendrite: p>0.32 for all frequencies; soma vs. null: p<0.0001 at>50 Hz, p<0.01 at 25 Hz, p>0.47 at<10 Hz; dendrite vs. null: p<0.0001 at>25 Hz, p<0.002 at 10 Hz, p=0.11 at 5 Hz. For somatic stimulation in (G): soma vs null: p<0.0001 for<50 Hz, p>0.44 for>100 Hz. For dendritic stimulation in (G): dendrite vs null: p>0.11 for all frequencies. For somatic stimulation in (H): soma vs null: p<0.0001 for 50 Hz; p>0.12 for all other frequencies. For dendritic stimulation in (H): dendrite vs null: p<0.02 for 5 Hz, p=0.10 for 10 Hz, p>0.48 for>25 Hz. GCaMP6s: n = 10 cells from 4 animals except for 5 Hz stimulation, where n = 5 cells from 3 animals; GCaMP6f: n = 9 cells from 3 animals.

Figure 4—figure supplement 1. Ex vivo calibration of GCaMP6s in soma and apical tuft dendrites across subthreshold and suprathreshold dendritic stimulation parameters.

(A) Experimental schemata. Layer 5 pyramidal neurons expressing GCaMP6s were recorded using whole-cell patch clamp in acute cortical slices of the primary visual cortex. The soma and distal apical dendrite (nexus) were imaged during layer 1 (L1) stimulation. Stimulation consisted of a train of 10 pulses delivered at 5–200 Hz and at a stimulus intensity 25–100% of somatic spike threshold. Negative deflections in electrophysiological traces are stimulation artefacts that have been cut for clarity. (B) Average somatic and dendritic calcium fluorescence across stimulation parameters. (C) Example calcium transients imaged in the soma and apical dendrite during 200 Hz stimulation at a range of stimulus intensities. Concurrent somatic electrophysiological recordings are shown below each transient. Note that stimulation conditions evoking low somatic output (i.e. single action potential) can trigger detectable calcium events in the dendrite that are not detected in the soma. (D) Average peak amplitude of calcium transients imaged in the soma (red) and dendrites (blue) plotted against evoked action potential number. Trials were binned by the number of action potentials (APs) evoked by stimulation. Dendritic calcium events are larger than somatic events in trials evoking a single action potential. Null distribution of peak amplitudes from sham stimulation trials are shown (dashed horizontal lines). (E) Proportion of compartment-specific events detected only in the dendrite and not in the soma, plotted against action potential number. Somato-dendritic coupling is high except when stimulation evokes a single somatic spike. (F) As in E but trials were binned by the magnitude of evoked dendritic calcium fluorescence instead of evoked action potential number. (G, H) As in D, E but trials were binned by evoked action potential frequency instead of number, for all trials with more than one action potential. Calcium signals in both soma and apical tuft were reliably detected across somatic spiking frequencies. For frequencies higher than 100 Hz, average calcium signal amplitude decreased owing to a decrease in number of action potentials generated by high frequency stimulation (within the relative refractory period). Significance was assessed with two way (D, G) and one way (E, F, H) repeated measures ANOVA with post-hoc Sidak tests. (D): soma vs. dendrite: p<0.001 at 1 AP; p<0.01 at>10 AP; p>0.23 for remaining conditions. (E): soma vs. dendrite: p<0.0001 at 1 AP; p>0.56 for all other conditions. (F) soma vs. dendrite: p<0.01 at 0.1 ΔF/F0; p>0.36 for all other conditions. (G) dendrite vs. soma p=0.63. (H) p=0.87. n = 10 cells from 4 animals except for 5 Hz stimulation, where n = 5 cells from 3 animals. Shaded areas represent S.E.M.