Using a linear array of 16 electrodes (Michigan probe), we recorded the extracellular local field potentials (LFPs) in the hindlimb area of the primary somatosensory cortex in the anesthetized rats, while optogenetically stimulating layer 5 (L5) of the cortex (Fig. 1a). Layer-specific light delivery was achieved with a micro-periscope consisting of a 0.18 mm x 0.18 mm micro right-angled prism, a custom-designed Grin lens, and a multi-mode optical fiber (Fig. 1b and Supplementary Fig. 1). Microinjection of adeno-associated virus conjugated with channelrhodopsin-2 (ChR2) and a CaMKIIα promoter to L5 was used to restrict expression of ChR2 predominantly to L5 pyramidal neurons16. The fluorescence image of the cortical slices taken from the recorded rats showed the confined viral expression in L5 (Fig. 1c).

Fig. 1 Optogenetically evoked dendritic Ca2+ spikes in the local field potential. a Schematic diagram of the experiment. b A photomicrograph of the micro-periscope system with collimated light in the air (top) and in the cortical tissue (bottom). c A cortical slice showing the virally transfected region co-expressing Channelrhodopsin 2 (H134R) and eYFP. Scale bar represents 500 μm. d Optogenetically evoked potentials at 16 cortical depths with the highest light intensity (12 mW/mm2), averaged over 100 traces in one animal. Shaded area indicates the late sink. Blue bar indicates the timing of L5 light stimulation. e Principal component analysis of 100 waveforms (top left) recorded at 600 μm below the pia (indicated by the arrow head in d) revealed three types of waveforms: those without discernable late spikes (bottom left); those with broad spikes (top right); those with huge-amplitude, sharp spikes (bottom right). f Current source density (CSD) analysis of the evoked potentials averaged over 100 measurements (left) and the expanded view between 300 and 900 μm below the pia showing that the late sink initiated at 600 μm below the pia and propagated both upward and downward. g The late sink depends on light intensity (one-way ANOVA, p < 0.001, n = 17 animals). Post hoc multiple comparison test indicates that the amplitude upon highest light intensity significantly differed from others (all p < 0.001). h Schematic diagram of Ca2+ fluorescence imaging experiments (left). The sharp increase in Ca2+ fluorescence measured in L2/3 coincided with the Ca2+ spikes measured with Michigan probe at 600 μm below the pia (right). Black and gray traces are the average of 10 representative measurements with and without discernable Ca2+ spikes respectively. The light intensity for L5 stimulation was highest (12 mW/mm2). i The late sink was unaffected by local application of the synaptic blocker CNQX (100 μM) in L2/3 and L5 (both p > 0.05, Wilcoxon signed rank test, n = 6). j Multi-unit activity (MUA) in L2/3 and L5 after optogenetic stimulation of L5 (averaged over n = 5 animals), at the highest light intensity (12 mW/mm2). Shaded area indicates the period when the late sink was evoked. Blue bar indicates the timing of L5 light stimulation. k The late sink significantly decreased by local application of 50 μM Baclofen in L2/3 (p < 0.05, Wilcoxon signed rank test, n = 5). l The late sink also significantly decreased by local application of 3 μM TTX in L5 (p < 0.05, Wilcoxon signed-rank test, n = 3) Full size image

A novel method to evoke dendritic Ca2+ spikes in vivo

Ca2+ spikes can be evoked in L5 pyramidal neurons by depolarizing the apical dendritic initiation zone17. Light pulses delivered to L2/3 caused a sink in these layers, however we chose not to use this approach because it was difficult to separate the effect of the ChR2 current from activation of voltage-sensitive postsynaptic currents. We therefore chose a second method for evoking dendritic Ca2+ spikes based on the “critical frequency” approach that uses high-frequency backpropagating action potentials (bAPs) generated at the soma18, 19. This method has been shown to activate the same dendritic Ca2+ channels as direct dendritic depolarization in vitro20 and has been shown to be effective in vivo21. By activating ChR2 in L5, we avoided the confounding influence on the LFP that would otherwise have occurred by direct activation of ChR2 in the dendrites (i.e., via light to L2/3). In other words, we could remotely activate the L5 dendrites without directly stimulating the apical dendrites either optically or synaptically.

A single pulse of light (10–30 ms) delivered to L5 reliably caused a current sink in L5 in all cases (Fig. 1d) but also, in 67.8 ± 23.7 (mean ± SD, n = 17 animals) % of trials with maximum light amplitudes, we observed an additional sink in the upper and middle layers at distances corresponding to the typical location of dendritic Ca2+ spikes8 ~ 20 ms after the offset of the light stimulation18. Interestingly, the current sink in L2/3 was reliably accompanied by a current source in L1 (Fig. 1d, f and Supplementary Fig. 2). The late sink in L2/3 was sometimes “simple”, and sometimes “complex” in form (Fig. 1e, right) and had variable amplitudes (0.48 ± 0.05 mV, n = 17 rats). In the remaining cases, there was no discernable late sink at all (Fig. 1e, bottom left). Current source density (CSD) analysis of the evoked potentials revealed that the second sink was initiated at 613 ± 27 μm below the pia and propagated in both directions, upward and downward (Fig. 1f). The sink was also highly dependent on stimulus strength with a sharp increase occurring at higher light amplitudes (p < 0.001, one-way ANOVA with post hoc multiple comparison, n = 17 animals, Fig. 1g and Supplementary Fig. 3).

The sink in L2/3 therefore bore all the hallmarks of a dendritic Ca2+ spike including location, timing and a sharp dependence on the strength of somatic activation. To investigate this possibility, we performed a number of tests. First, we repeated the optogenetic stimulation experiments combined with a second micro-periscope for recording Ca2+ fluorescence in L2/3 (Fig. 1h, left). For these experiments, a Ca2+ indicator (Cal-590 AM) with a long excitation wavelength (550 nm) to avoid direct activation of ChR2 was injected to L5. The light through this micro-periscope was shone tonically throughout the experiment so that any residual effect was not time locked to the ChR2 stimulation in L5. Here, optogenetic activation of L5 caused an all-or-none fluorescence transient in L2/3 (at higher stimulus strengths) similar to the current sink seen earlier (Fig. 1h, right). Here the term all-or-none is used to describe the fact that the late sink, while it varied in amplitude and duration, was entirely absent in a significant number of cases resulting in a bimodal amplitude distribution (Supplementary Fig. 4). This is consistent with the late sink correlating with a thresholded event such as a dendritic Ca2+ spike. This Ca2+ transient was dendritic because the indicator, applied in L5, could only have reached L2/3 by diffusing along the apical dendrites of L5 cells22, 23 which suggested that the current sink seen earlier also corresponded to dendritic Ca2+ currents. However, to rule out contribution from other postsynaptic elements such as disynaptic firing of cells in L2/3 we injected 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 100 μM) into L2/3 and L5. In these experiments, there was no change in the amplitude of the current sink in L2/3 (both p > 0.05, Wilcoxon signed rank test, Fig. 1i and Supplementary Fig. 3). We also measured multi-unit activity (MUA) in both L2/3 and L5 (Fig. 1j), which did not show any significant increase of MUA before the onset of the late sink compared with the prestimulus period (all p > 0.05, Student’s paired t-test, n = 5 animals). We therefore conclude that the late current sink in L2/3 was produced by currents in the dendrites of L5 pyramidal neurons.

Previous studies have shown that dendritic Ca2+ spikes are effectively blocked by the GABA B receptor agonist baclofen24, 25 due to down-regulation of Ca v 1 (L-type) channels12. Here, we found that the dendritic current sinks significantly decreased by local injection of 50 μM baclofen to L2/3 (from 0.39 ± 0.05 to 0.09 ± 0.01 mV, n = 5, p < 0.05, Wilcoxon signed rank test) (Fig. 1k and Supplementary Fig. 5). Local injection of 3 μM TTX into L5 which would be expected to block APs in the L5 pyramidal neurons also significantly decreased the dendritic current sinks (from 0.43 ± 0.06 to 0.11 ± 0.02 mV, n = 3, p < 0.05, Wilcoxon signed rank test; Fig. 1l and Supplementary Fig. 6). The late dendritic current sink was clearly not due to bAPs in the dendrites, because they would be expected to take only 1.5 ms to propagate 1 mm back along the dendrites in vivo at 0.67 m/s14. Taken together, we conclude that the current sink observed in the middle to upper layers was due to dendritic Ca2+ spikes in L5 pyramidal neurons.

A Ca2+ spike-evoked passive current source in L1

The appearance of a passive current source in the superficial layers correlating with the Ca2+ spike sink in L2/3 was an unexpected finding. Several studies have observed that Ca2+ spikes originate in the apical trunk dendrite and rarely propagate to the most distal tuft branches8, 10, 13, 26, 27. Furthermore, the distal tuft branches have a high density of the non-selective cation channel (I h )19, 28,29,30 that is spontaneously open, and previous in vitro studies showed that I h interacts with dendritic Ca2+ spikes31,32,33. This spontaneous leak current may account for the pronounced source of passive current. We tested this possibility by local application of I h blocker ZD7288 (500 μM) on the cortical surface (Fig. 2a). After ZD7288 application the second sink was elongated upward and the source was significantly reduced in both size and duration (all p < 0.05, Wilcoxon signed rank test, Fig. 2b, c), suggesting that I h enhanced the passive current under control conditions. By similar reasoning, we hypothesized that the source near the cortical surface produced by currents in the distal tuft dendrite should also lead to positively deflected potentials at the cortical surface. Using a 4 by 4 surface electrode array (Fig. 3a), we detected surface positive potentials that were 20–30 ms delayed after the onset of the dendritic Ca2+ spikes measured at 600 μm deep below the pia (Fig. 3b). In all animals (n = 4) the evoked surface potentials were clearly detected at multiple electrodes that were 500 μm apart. The surface potentials were also baclofen-sensitive (from 0.39 ± 0.13 to 0.01 ± 0.03 mV, n = 4, p < 0.05, Wilcoxon signed rank test, Fig. 3c) and clearly detectable even in the awake freely moving animals (n = 3) with similar characteristics (Fig. 4a–c, all p > 0.05, Wilcoxon rank sum test). We conclude that apical dendritic Ca2+ spikes are detectable as positive potentials at the cortical surface in both anesthetized and awake brain.

Fig. 2 I h contributes to the generation of passive current in L1. a Schematic diagram of the experiment. b CSD profiles of evoked potentials before (left) and after application of 500 μM ZD7288 to the cortical surface (right), averaged over 100 measurements. c Summary of the CSD amplitude at 100 to 300 μm below the pia before and after ZD7288 application at the cortical surface (all p < 0.05, Wilcoxon signed-rank test) Full size image

Fig. 3 Optogenetically evoked dendritic Ca2+ spikes greatly affect surface potentials. a Schematic diagram of the experiment. Surface potentials recorded at the electrode close to the micro-periscope (marked in red) were analyzed. b Surface potentials generated by optogenetically evoked Ca2+ spikes (top) aligned with LFPs recorded at 600 μm below the pia (bottom). Black and gray traces (average of 10 measurements) are with and without discernable Ca2+ spikes, respectively. c Average surface potentials before and after application of 50 μM baclofen into L2/3 (n = 4, p < 0.05, Wilcoxon signed-rank test) Full size image

Fig. 4 Optogenetically evoked surface potentials in the awake brain. a In agreement to numerous previous studies, the surface potential recorded in the awake brain shows high-frequency components compared with the anesthetized brain. b Average trace of 50 measurements of surface potential with (black) and without the late spike (gray). Blue bar indicates the timing of L5 light stimulation. c Summary of characteristics in the anesthetized and awake brain (all p > 0.05, Wilcoxon rank sum test, 4 anesthetized animals vs 3 awake animals) Full size image

Sensory-evoked dendritic Ca2+ spikes

Having determined the impact of dendritic Ca2+ spikes on surface potentials in the absence of synaptic input to the dendrite (i.e. evoked by optogenetic stimulation of L5), we turned our attention to the effect of sensory-evoked Ca2+ spikes on surface potentials. We recorded LFPs, surface potentials, and Ca2+ fluorescence in the same cortical area—the hindlimb area of the primary somatosensory cortex—while electrically stimulating the contralateral hindlimb (Fig. 5a). Simultaneous recording of LFPs and Ca2+ fluorescence revealed two distinct patterns of activity in response to hindlimb stimulation (Fig. 5b). The first kind of response showed a current sink across L2/3 and L5 approximately 10 ms after the stimulus onset, which corresponded to excitatory postsynaptic potentials (EPSPs) as shown in previous studies34,35,36. The first current sink was correlated with a small Ca2+ transient observed with the dendritic micro-periscope (Fig. 5e, left). The second type of response arrived ~ 50–60 ms after the stimulus and showed a second current sink in the middle and uppers layers with a similar amplitude and timing to the late Ca2+ spike-evoked sink produced by optogenetic stimulation of L5 (Fig. 5b, right). Moreover, the sensory-evoked late sink was accompanied by a large increase in dendritic Ca2+ signal (Fig. 5c, e) also similar in amplitude and duration to the optogenetically evoked dendritic Ca2+ transient. A principal component analysis of the LFP waveform (Fig. 5d) revealed that the second type of response could be further partitioned into simple or complex waveforms, with the complex waveforms accompanying larger dendritic Ca2+ transients (Fig. 5e, middle & right). Consistent with optogenetically evoked dendritic Ca2+ spikes, the second current sink was initiated at 538 ± 57 μm below the pia, propagated in two directions (Fig. 5f, right), and generated a passive current source in the superficial layers (Fig. 5f, middle). Moreover, in all animals (n = 8) this second current sink was abolished by local injection of baclofen in L2/3 (from 0.50 ± 0.12 to 0.00 ± 0.05 mV, p < 0.05, Wilcoxon signed rank test, Fig. 5g) while the first current sink did not significantly change (from 0.73 ± 0.12 to 0.43 ± 0.07 mV, p > 0.05, Wilcoxon signed rank test, Supplementary Fig. 7). Lastly, the surface positive potentials passively generated by the second current sink were clearly detectable with the surface electrode array (Fig. 6a, b). Many of these surface potentials were as large as those generated by the first current sink which was presumably due to synaptic input (Fig. 6b, c), although the average amplitude of the second current sink was smaller than the first (first: 0.71 ± 0.01 vs second: 0.59 ± 0.02 mV, n = 6 rats, p < 0.001, Wilcoxon rank sum test). Moreover, the second surface spike was significantly wider than the first (p < 0.05, Wilcoxon signed rank test, n = 6 animals, Fig. 6d, left) and importantly, optogenetically evoked surface potentials had a similar width (p > 0.05, Wilcoxon rank sum test, Fig. 6d, left). The peak latency of the second spike was also similar to that of the optogenetically evoked surface potential (p > 0.05, Wilcoxon rank sum test, Fig. 6d, right). Two-photon Ca2+ imaging of individual dendrites of L5 pyramidal cells (Fig. 7a–c) and simultaneous recording of surface potentials further revealed the strong correlation between the amplitude of the second surface potential and the peak fluorescence change (r = 0.7693, p < 0.01, n = 38 dendrites from 4 animals, Fig. 7d, e). To examine the predictive power of our characterization, we developed a simple classifier using half of the paired measurements of two-photon dendritic Ca2+ fluorescence and simultaneously recorded surface potentials (the “training” data); then we tested the classifier with the remaining half (the “validation” data, Fig. 7f, see Methods section for more details). Simply taking the average surface potential 50–60 ms after stimulus onset and setting a lower threshold θ 1 that corresponds to the upper limit of fluorescence noise (mean M 0 + one standard deviation SD 0 when surface potentials are zero), the classifier predicts that the surface potentials correspond to Ca2+ spikes with 73.9% (34/46) accuracy (light gray area in Fig. 7f). With a higher threshold θ 3 that corresponds to M 0 + 3SD 0 , the classifier predicts Ca2+ spikes with 100% (10/10) accuracy (dark gray area in Fig. 7f). Taken together, we conclude that sensory stimulation evokes EPSPs that can be detected ~ 10 ms after the stimulation followed in some cases by dendritic Ca2+ spikes in L5 pyramidal neurons that have a comparable impact at the cortical surface in the form of positive potentials arriving ~ 50–60 ms after stimulation.

Fig. 5 Sensory-evoked dendritic Ca2+ spikes. a Schematic diagram of the experiment. b Sensory-evoked potentials with or without late sink. The late sink was considered present if the peak amplitude exceeded 3× s.d. of the prestimulus activity. Shaded area indicates the late sink. The late sink was present in all rats (n = 8). c Correlation between the LFP amplitude of the late sink at 600 μm below the pia and dendritic Ca2+ transients (p < 0.05 in all animals). d Principal component analysis of 100 waveforms recorded at 600 μm below the pia (indicated by arrow head in b) in a representative animal. e Simultaneously recorded LFPs (top) and Ca2+ transients (bottom). From left to right: those without discernable late sink; those with smaller late sink; those with larger late sink (from part d). f CSD profiles of sensory-evoked potentials with (left) or without (middle) the late sink (from part b) and the expanded view of the CSD profile between 300 μm and 700 μm showing that the late sink was initiated at 500 μm below the pia and then propagated both upward and downward (right). g The late sink (black, average over all 100 traces) was abolished by 50 μM baclofen (magenta, average of 100 traces) shown in a single case (top) and for all rats (bottom; p < 0.05, Wilcoxon signed-rank test, n = 8) Full size image

Fig. 6 Sensory-evoked dendritic Ca2+ spikes also greatly affect surface potentials. a Schematic diagram of apparatus for recording sensory-evoked surface potentials. b Surface potentials aligned with LFPs recorded at 600 μm below the pia. Thick lines denote the average over 10 measurements; thin lines show representative traces where the late sink is as large as the first. c Surface positive potentials evoked by the first sink (top) and those evoked by the late sink (bottom). Pooled data from all rats (n = 6). d Width and peak latency of the first and second spikes evoked by sensory stimulus, compared to optogenetically evoked late spike Full size image