Decision letter: Frequency- and spike-timing-dependent mitochondrial Ca2+ signaling regulates the metabolic rate and synaptic efficacy in cortical neurons (2021)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Mitochondrial activity is crucial for the plasticity of central synapses, but how the firing pattern of pre- and postsynaptic neurons affects the mitochondria remains elusive. We recorded changes in the fluorescence of cytosolic and mitochondrial Ca2+ indicators in cell bodies, axons, and dendrites of cortical pyramidal neurons in mouse brain slices while evoking pre- and postsynaptic spikes. Postsynaptic spike firing elicited fast mitochondrial Ca2+ responses that were about threefold larger in the somas and apical dendrites than in basal dendrites and axons. The amplitude of these responses and metabolic activity were extremely sensitive to the firing frequency. Furthermore, while an EPSP alone caused no detectable Ca2+ elevation in the dendritic mitochondria, the coincidence of EPSP with a backpropagating spike produced prominent, highly localized mitochondrial Ca2+ hotspots. Our results indicate that mitochondria decode the spike firing frequency and the Hebbian temporal coincidences into the Ca2+ signals, which are further translated into the metabolic output and most probably lead to long-term changes in synaptic efficacy. Introduction For the neuronal circuit to function properly, the energy demand in all compartments of the individual neurons needs to be precisely matched by local, primarily mitochondrial (Celsi et al., 2009; Rangaraju et al., 2019), ATP production. During periods of enhanced neuronal activity, mitochondria accelerate ATP production by allowing the cytosolic Ca2+ elevations to propagate into the mitochondrial matrix (Ashrafi et al., 2020; Díaz-García et al., 2021). When cytosolic [Ca2+]i rises, Ca2+ ions, powered by the steep mitochondrial membrane potential, flow into the mitochondrial matrix via the Ca2+ uniporter MCU (Baughman et al., 2011; De Stefani et al., 2011), and are then extruded back into the cytosol by the mitochondrial Na+/Ca2+ antiporter NCLX (Palty et al., 2010). The mitochondrial Ca2+ elevation increases ATP production by activating at least three Krebs cycle enzymes (De Stefani et al., 2016; Wescott et al., 2019). When Ca2+ signaling is disrupted, mitochondria in presynaptic terminals fail to maintain the stable ATP concentration during enhanced activity periods (Ashrafi et al., 2020; Giorgio et al., 2013; Glancy and Balaban, 2012). The link between the distinctive types of the neuronal electrical activity, mitochondrial Ca2+ signaling and metabolism in other neuronal compartments is poorly understood, however. Recent evidence using photo-uncaging of glutamate on spines of cultured hippocampal neurons indicates that dendritic mitochondria play a critical role in long-term regulation of synaptic strength (Rangaraju et al., 2019). Thus, local inhibition of the mitochondria by a phototoxic protein abolishes the synaptic plasticity in the affected dendritic segment. Under physiological conditions, the plastic changes in synaptic efficacy are believed to be controlled by the relative timing of pre- and postsynaptic action potentials (APs) (Bi and Poo, 1998; Holtmaat and Svoboda, 2009; Markram et al., 1997b). Spike-timing–dependent plasticity (STDP) relies on activation of the postsynaptic NMDA receptors, which are Ca2+ permeable and require both glutamate and depolarization to open (Nowak et al., 1984). Mitochondrial handling of Ca2+ ions is known to have a significant effect on cytosolic Ca2+ dynamics (Rizzuto et al., 2004; Szabadkai and Duchen, 2008). The mitochondrial depletion, however, had no effect on glutamate evoked cytosolic Ca2+ dynamics (Rangaraju et al., 2019), indicating that these organelles play a yet unexplored downstream role in the cascade of reactions leading to STDP. Here, using whole-cell electrical recordings from Layer 5 pyramidal neurons in cortical slices and fluorescence imaging of cytosolic and mitochondrial Ca2+ indicators, we show that single or few spikes trigger rapidly rising and decaying mitochondrial Ca2+ elevations in all neuronal compartments, with kinetics similar to cytosolic Ca2+ transients. Our evidence indicates that the mitochondria's Ca2+ signaling and metabolic rate depend critically on spike firing frequency. We further report that, in dendrites, the coincidence of unitary EPSP and a backpropagating action potential produces a localized [Ca2+]m transient, which, in addition to enhancing local ATP synthesis, could play a role in STDP. Results Spike-elicited mitochondrial Ca2+ transients To determine how neuronal electrical activity affects mitochondrial Ca2+ dynamics, we performed somatic whole-cell recordings from L5 pyramidal neurons expressing the mitochondria-targeted, neuron-specific Ca2+ indicator, mitoGCaMP6m. The intracellular solution was supplemented with the cytosolic Ca2+ indicator, Fura-2, that could be excited separately from mitoGCaMP6m. Series of thin, high-resolution optical sections over the vertical extent of the neuron, representing Fura-2 and mitoGCaMP6m fluorescence elicited by two-photon excitation at 760 and 960 nm, respectively, were used to reconstruct the morphology of the neuron and to reveal the position of the individual mitochondria within its soma and processes. To improve the temporal resolution of the optical signals, dynamic fluorescence measurements were obtained from the smaller regions of interest in soma, axon, and dendrites. In a typical experiment, two APs elicited by the injection of two brief current pulses via the patch pipette caused cytosolic and mitochondrial Ca2+ elevations in the soma of an L5 cell (Figure 1a). Although the cytosolic Ca2+ signals had relatively even intensity, the change in mitoGCaMP6m fluorescence occurred at 'hotspots', each representing an individual mitochondrion. The mitochondrial Ca2+ elevations began with a short delay after the beginning of the spike train, and they were observed only in the electrically active neurons. At the same time, the fluorescence of the nearby mitoGCaMP6m expressing mitochondria belonging to the non-active cells did not change (Figure 1—figure supplement 1). Figure 1 with 6 supplements see all Download asset Open asset Trains of action potentials elicit mitochondrial Ca2+ elevations in soma and processes of L5 pyramidal neurons. (a) Changes in mitoGCaMP6m and Fura-2 fluorescence elicited by a train of two APs in a representative L5 pyramidal cell. Left, The image obtained by merging two optical sections through part of a L5 neuron at excitation wavelengths of 760 and 960 nm, eliciting the Fura-2 and mitoGCaMP6m fluorescence, respectively. Right, Somatic mitoGCaMP6m (red) and Fura-2 (green) ΔF/F transients elicited by a train of two APs. MitoGCaMP6m trace (red) is an ensemble average of 16 consecutive sweeps. (b) Small-amplitude mitoGCaMP6m transients decay rapidly. Left, Localization of the mitoGCaMP6m labeled mitochondria in a representative L5 neuron. The image was obtained by merging the fourteen, 1 µm thick optical sections at excitation wavelengths of 760 and 960 nm, eliciting the Fura-2 and mitoGCaMP6m fluorescence, respectively. The yellow color indicates colocalization of the Fura-2 (green) and mitoGCaMP6m (red) fluorescence, revealing the position of the mitochondria. The rectangle indicates the region of interest from which the optical sweeps were obtained. Right, somatic mitoGCaMP6m (red) and Fura-2 (green) transients elicited by 5, 20, and 50 APs in a cell shown on the left. Black lines are the best single or double exponential fits of the decay. Notice that after the relatively small [Ca2+]m elevations evoked by five or 20 APs, the Ca2+ clearance is rapid and follows the single exponential time course (τ ~ 0.7 s). The decay of larger transients evoked by 50 APs is biexponential (τ1 of ~0.7 s and τ2 of ~28 s). (c) Decay time course of mitochondrial Ca2+ transients depends on their amplitude. Each dot represents a decay time constant of the mitoGCaMP6m transient (τ1 for monoexponential, τ1 and τ2 for bi-exponential decays) obtained in recordings from 42 neurons and plotted against its peak amplitude. Continuous lines are mean τ1 (n = 55, black) and τ2 (n = 23, red); dashed lines represent standard deviation from the mean. Arrow represents the amplitude of the smallest mitoGCaMP6m transient (ΔF/F = 44 %), which decayed biexponentially. (d) The amplitude of spike-evoked mitoGCaMP6m fluorescence transients varies between different neuronal processes. Left, Localization of the mitoGCaMP6m labeled mitochondria in a representative L5 pyramidal neuron. The rectangles indicate the regions within the apical dendrite (cyan), basal dendrite (magenta), and axon initial segment (red) from which fluorescence measurements were obtained. Right, Mitochondrial and cytosolic (green) Ca2+ transients elicited by a train of 5 APs at 50 Hz in different neuronal compartments. (e) Mean peak ΔF/F of the mitoGCaMP6m transients as a function of the number of action potentials in soma, apical, and basal dendrites, and axon initial segment. Shown are mean values ± SE (n = 5–29). The AP-elicited mitochondrial Ca2+ elevations required Ca2+ influx from the extracellular space. It is likely that Ca2+ ions enter via the voltage-gated Ca2+ channels, as bath application of Cd2+ (200 µM) inhibited both cytosolic and mitochondrial Ca2+ transients (Figure 1—figure supplement 2). Previous studies have shown that Ca2+ depletion of the endoplasmic reticulum (ER) in cultured neurons does not influence mitochondrial Ca2+ elevations (Ashrafi et al., 2020). Consistent with these results, we found that blockade of ER Ca2+ channels by Ryanodine (100 µM) and Dantrolene (100 µM) (Figure 1—figure supplement 3) had no significant effect on mitochondrial Ca2+ transients. An increase in the number of spikes produced progressively larger mitoGCaMP6m transients in the soma (Figure 1b) and the apical dendrite (Figure 1—figure supplement 4) with a progressively slower rising phase. Thus, the mito-Ca2+ signals elicited by 50 APs grew throughout the spike train duration, reaching a peak ΔF/F value of 59% ± 11% (n = 29) and 54% ± 11% (n = 22) for soma and apical dendrite, respectively, at 120 ± 18ms (n = 10) after the train end. In contrast with previous reports (Ashrafi et al., 2020; Lin et al., 2019), mito-Ca2+ transients elicited by 2–20 spikes decayed as rapidly or even faster than cytosolic Ca2+ transients. However, following large elevations, [Ca2+]m remained high for tens of seconds, reaching the resting level long after the cytosolic Ca2+ concentration completely recovered. We systematically examined the relationship between the peak amplitude and the decay rate of the mito-Ca2+ transients in 42 pyramidal neurons (Figure 1c, Figure 1—figure supplement 5). Decay of smaller transients (peak ΔF/F < 40%) was always monoexponential, with τ = 0.86 ± 0.11 s (n = 25) as well as decay of some middle-sized (ΔF/F 40–75%) transients (τ = 0.82 ± 0.10 s, n = 10). The decay of other middle size and large (ΔF/F > 75%) transients was double exponential, with τ1 = 0.86 ± 0.13 s and τ2 = 22.0 ± 2.85 s (n = 23). The decay of cytosolic Ca2+ transients always followed a single exponential time course which was characterized by τ = 1.03 ± 0.14 s (n = 22) for small and τ = 1.19 ± 0.4 s (n = 20, p = 0.43) for large transients. To elucidate the differences in mitochondrial Ca2+ signaling between distinct neuronal compartments, we monitored mitoGCaMP6m fluorescence in ~10 µm long regions of interest in basal, apical dendrites, and axon initial segments (AISs) during trains of five APs (Figure 1d). While the amplitude and time course of the cytosolic Ca2+ responses in all these compartments were similar, the magnitude of mitochondrial signals was remarkably polar. The amplitude of the mitochondrial Ca2+ elevations in apical dendrite was as prominent as in the soma. In contrast, in the AIS and thin basal dendrites, the mito-Ca2+ responses were dramatically smaller. We next sought to evaluate the relationship between the number of APs and the mean peak amplitude of the mitoGCaMP6m transients in the soma, AIS, apical and basal dendrites of 29 neurons (Figure 1e). The steepness of this relationship was greater in soma and apical dendrites (1.3% and 1.1% ΔF/F per spike, respectively) compared with AIS and basal dendrites (0.4% and 0.3% ΔF/F per spike, respectively). The compartmental differences in mitochondrial signals were not due to a different magnitude of cytosolic Ca2+ elevations. The pattern of [Ca2+]i during the neuronal activity is known to be complex. However, the differences in peak cytosolic Ca2+ levels were subtle, and they poorly correlated with the magnitude of mitochondrial Ca2+ elevations. For example, the peak ΔF/F amplitude of Fura-2 transients elicited by twenty spikes was 26% ± 3.6% (n = 22) for soma, 37% ± 4% (n = 13) for apical dendrite, 38% ± 4% (n = 13) for basal dendrites, and 25% ± 4% in the AIS (n = 7). The compartmental differences in magnitude of the mitoGCaMP6m transients could, at least partially, be explained by the lower expression level of fluorescence probe in the mitochondria localized within the thinner neuronal processes. This seems to be unlikely, however, since, in all neuronal compartments, maximal mitoGCaMP6m fluorescence of the individual mitochondria following prolonged depolarization of the neuronal membrane was similar (Figure 1—figure supplement 6). Our evidence, therefore, points to the existence of an as-yet-unidentified mechanism that differentially regulates the mitochondrial Ca2+ entry in distinct neuronal compartments. Frequency-dependent amplification of mitochondrial Ca2+ elevations Next, we examined whether mitochondrial Ca2+ elevations are sensitive to firing frequency. Figure 2a shows an optical recording from the soma of a representative L5 neuron in which cytosolic and mitochondrial Ca2+ transients were elicited by trains of twenty APs at 20, 50, and 100 Hz. As spike frequency increased, the rise of cytosolic Ca2+ transients became progressively faster and their peak amplitude modestly increased (Figure 2—figure supplement 1). In contrast, the mitochondrial Ca2+ signals showed a very different frequency dependence. Firing at a frequency of 20 Hz or lower elicited a minimal elevation in [Ca2+]m, whereas the response to spikes at a frequency of 50 Hz or higher was dramatically greater. The steep frequency dependence of the mitochondrial signals was observed in all neuronal compartments, including somas, apical, and basal dendrites, making it unlikely that it reflects the frequency-dependent failure of AP backpropagation (Spruston et al., 1995). The frequency-dependent amplification of mitochondrial Ca2+ transients was observed in all 21 neurons tested with either 50, 20, or 5 APs (Figure 2b). The mean ratio of peak amplitudes of the mitochondrial Ca2+ transients elicited by trains of spikes at 50 and 20 Hz was larger when neurons were subjected to longer (3.36 ± 0.46 times, n = 22 for 50 APs) than to shorter (1.74 ± 0.18 times, n = 5 for 5 APs) trains (Figure 2c). Systematic varying of AP frequency in a range from 10 to 100 Hz revealed that the peak amplitude of mito-Ca2+ transients behaved as Bolzmannian function of the frequency, with mean half-amplitude of 38 Hz and steepness of 22 Hz–1 (n = 12). Figure 2 with 2 supplements see all Download asset Open asset Frequency-dependent amplification of the mitochondrial Ca2+ elevations. (a) Cytosolic and mitochondrial Ca2+ elevations elicited at the soma of a representative pyramidal neuron by trains of 20 APs at 100, 50, and 20 Hz. Left, The top image is mitoGCaMP6m labeled mitochondrial map of a representative L5 pyramidal neuron, obtained by merging the Fura-2 and mitoGCaMP6m fluorescence (for detail, see Figure 1). The bottom image is the maximum intensity Z-projection representing the Fura-2 fluorescence only. Right, The mitoGCaMP6m (red) and Fura-2 (green) ΔF/F transients elicited at the soma by the spike trains at indicated frequency. Notice that, at 20 Hz, the train of spikes produced almost no mitochondrial Ca2+ elevation, while the amplitude of the cytosolic Ca2+ transient changed little as a function of spike frequency. (b) Peak amplitude of mitoGCaMP6m (red) and Fura-2 (green) ΔF/F transients elicited by a train of 50 (top) or 20 (bottom) APs at a frequency of 20 and 50 Hz. The gray lines connect the paired values obtained from the same individual neuron at two firing frequencies. Box plots represent the 25–75% interquartile range, and the whiskers expand to the 5–95% range. A horizontal line inside the box represents the median of the distribution, and the mean is represented by a cross symbol (X). (c) Mean ratio of peak amplitudes of mitoGCaMP6m (red) and Fura-2 (green) ΔF/F transients elicited by trains of 50, 20, and 5 APs at 50 and 20 Hz frequency. The decay time course of the mitochondrial Ca2+ transients elicited by high-frequency spike trains was significantly slower than those produced by the same number of spikes at a lower frequency (Figure 2—figure supplement 2). Thus, trains of fifty APs at 50 Hz elicited the extremely slowly decaying mito-Ca2+ transients (τ > 10 s) in 11/15 somas and 7/10 apical dendrites. In contrast, all but one mito-Ca2+ transient produced in the same neurons by fifty APs at 20 Hz decayed rapidly. Frequency-dependent acceleration of the mitochondrial NAD(P)H metabolism To determine whether the frequency-dependent amplification of Ca2+ signals in the mitochondria affects their metabolic activity, we monitored the changes in NAD(P)H autofluorescence elicited by trains of brief, just suprathreshold antidromic stimuli at a different frequency (Figure 3a). Whole-cell recording from a single pyramidal neuron within the region of interest was obtained to tune the stimuli intensity such that each stimulus would elicit only one AP. In cortical neurons, NAD(P)H signals primarily reflect changes in mitochondrial NAD(P)H pool (Díaz-García et al., 2021). In response to electrical stimulation, we observed a negative deflection in the NAD(P)H autofluorescence ('dip') which indicates an increased rate of electron transfer reflected in NAD(P)H oxidation, followed by a positive transient ('overshoot') which indicates Krebs-cycle-dependent replenishment of NAD(P)H pool. At higher stimulation frequency, the magnitude of both dip and overshoot of the NAD(P)H signals were enhanced, consistent with the previous reports that the rates of NAD(P)H oxidation and synthesis are dependent on the Ca2+ level in the mitochondrial matrix (Díaz-García et al., 2021). Spatio-temporal analysis of the NAD(P)H autofluorescence dynamics at two stimulation frequencies (Figure 3b) revealed that the changes in the fluorescence were spatially restricted to Layer 5 of a single cortical column and that the frequency-dependent amplification of both dip and overshoot amplitude was relatively uniform within the stimulated region. A comparison of NAD(P)H autofluorescence changes in response to a train of 50 stimuli at 20 Hz and 50 Hz in ten cortical slices (Figure 3c) revealed the same frequency dependence as with [Ca2+]m. The dip amplitude increased from –15 ± 2 a.u. at 20 Hz to –21 ± 2 a.u. at 50 Hz (n = 15 ROIs, p < 0.001), the overshoot's peak amplitude increased from 8 ± 2 a.u. to 19 ± 3 a.u. (p < 0.001) and the overshoot area increased from 156 ± 46 a.u.∙s to 441 ± 71 a.u.∙s (p < 0.001), respectively. Because the glial responses might partially contaminate NAD(P)H signals obtained in the cortical slices, we tested the frequency dependence of NAD(P)H responses in the in Stratum pyramidale of CA1 area of the hippocampus (Figure 3—figure supplement 1) which predominantly contains neuronal cell bodies. As in the neocortex, NAD(P)H signals in response to trains of stimuli delivered to the Stratum oriens were significantly enhanced at the higher stimulation frequency. Figure 3 with 1 supplement see all Download asset Open asset Frequency-dependent amplification of spike elicited changes in mitochondrial NAD(P)H auto-fluorescence. (a) In a representative cortical slice, changes in NAD(P)H fluorescence in response to extracellular stimuli trains depend on stimulation frequency. Left, DIC image of a coronal slice during the electrical and optical recording. The rectangle indicates the region from which the auto-fluorescence measurements were obtained. The stimuli were delivered via the bipolar electrode placed on the white-gray matter border, and the whole-cell recording was obtained from an L5 neuron within the same cortical column. Right, The membrane potential and optical traces evoked by trains of 50 just suprathreshold stimuli at 50 Hz (black) and 20 Hz (red). Notice that both dip and overshoot of the NAD(P)H signals are more prominent at 50 Hz. Inset: Stimuli intensity was carefully adjusted to elicit only a single AP per stimulus. (b) The amplitude of NAD(P)H signals depends on stimulation frequency, whereas their spatial extent does not. Shown are pseudocolor maps of change in the NAD(P)H fluorescence between the times by the in (c) frequency stimulation an increase in the magnitude of the dip and of the overshoot of the NAD(P)H Box plots representing the of the dip the of the overshoot and the area of the overshoot of the NAD(P)H signals evoked by trains of 20 Hz (red) and 50 Hz (black) The gray lines connect the paired values obtained from the same cortical regions at two firing frequencies (n = 15 ROIs, ten cortical slices, three Box plots represent the 25–75% interquartile range, and the whiskers expand to the 5–95% range. A horizontal line inside the box represents the median of the distribution, and the mean is represented by a cross symbol (X). dendritic [Ca2+]m elevations elicited by the coincidence of postsynaptic AP and EPSP We next sought to elucidate how synaptic activity affects mitochondrial Ca2+ the cell for to the of Fura-2 into the dendrites, we the bipolar electrode to an apical or basal of a single brief stimulus with its amplitude adjusted to the EPSP the for postsynaptic spike produced no detectable cytosolic or mitochondrial Ca2+ signals (n = 7). The single AP elicited by brief somatic current however, produced a cytosolic but no mitochondrial Ca2+ response in the dendrites. the coincidence of the EPSP and backpropagating AP to elicit a cytosolic and mitochondrial Ca2+ response (Figure While most dendritic mitochondria were the EPSP and AP coincidence single localized mitochondrial Ca2+ with peak ΔF/F amplitude of ± (n = in the dendritic regions of We the of these as evidence for a highly probably single mitochondrial Ca2+ elevation, in the of the active The unitary of the mitochondrial signals this be explained by a spatial of the by the presynaptic on the dendrites of the postsynaptic cortical cell et al., such that only one of a few active spines could be found in the relatively short segment of a dendritic that we The cytosolic Ca2+ elevation, in the same at which the mitoGCaMP6m was was not significantly larger than in the nearby The failure to see the cytosolic Ca2+ most due to temporal and amplitude resolution of optical recording that was to reveal Ca2+ elevation in the between the and the during the fast single Ca2+ transient and et al., We analysis by the cytosolic and mitochondrial Ca2+ signals elicited by 20 APs and APs paired with In both apical and basal dendrites, the paired APs produced a significantly larger mitochondrial In contrast, the cytosolic Ca2+ elevation amplitude was not significantly different for and paired stimulation (Figure and the peak amplitude of the mitoGCaMP6m transients elicited by paired APs was ± (n = and ± (n = times higher than of those evoked by the APs in the basal and apical dendrites, (Figure The of postsynaptic neurons to an AP was crucial for the dendritic mito-Ca2+ transients. Thus, intracellular with a solution a of voltage-gated channels, which the firing of the postsynaptic cells and while only a effect on the EPSP dramatically the amplitude of evoked mito-Ca2+ transients (Figure supplement 1). As with single even the significantly larger evoked in neurons by a train of synaptic in the of produced no significant Ca2+ elevation in dendritic mitochondria. Figure with 2 supplements see all Download asset Open asset The coincidence of postsynaptic action potential and EPSP localized mitochondrial Ca2+ elevations in the dendrites. (a) potential elicited by a just suprathreshold synaptic stimulus a spatially restricted increase in dendritic mitoGCaMP6m fluorescence, whereas action potential evoked by a 5 current cell had no such Mitochondrial map in a representative L5 pyramidal neuron, obtained by merging the Fura-2 and mitoGCaMP6m fluorescence (for detail, see Figure 1). The rectangle indicates the region within the apical dendrite from which fluorescence measurements were obtained. maps of change in the mitoGCaMP6m and Fura-2 fluorescence in response to a and current evoked AP. The mitoGCaMP6m (red) and Fura-2 (green) ΔF/F transients from regions of interest as indicated in the and the somatic membrane potential Arrow indicates a at which the synaptic stimulus elicited a mitochondrial Ca2+ The transients are ensemble of 50 sweeps. (b) potentials elicited by a train of synaptic stimuli larger mitoGCaMP6m signals in the dendrites than APs elicited by the injection of a train of current map in a representative L5 pyramidal neuron, obtained by merging the Fura-2 and mitoGCaMP6m The rectangles indicate the regions within the apical and basal dendrites from which fluorescence measurements were obtained. Right, of ΔF/F mitoGCaMP6m transients elicited in the apical (red) and basal dendrite by twenty suprathreshold synaptic stimuli at 50 Hz and by twenty brief current pulses delivered to the soma. The and traces are ΔF/F Fura-2 transients in apical and basal dendrites, respectively. The traces represent somatic membrane (c) Peak amplitude of mitoGCaMP6m and Fura-2 ΔF/F transients elicited in basal (top) and apical dendrites (bottom) by a train of twenty suprathreshold current pulses or synaptic stimuli at a frequency of 50 Hz. The line the paired values obtained from the same individual neuron at two stimulation Box plots represent the 25–75% interquartile range, and the whiskers expand to the 5–95% range. A horizontal line inside the box represents the median of the distribution, and the mean is represented by a cross symbol (X). (d) Mean ratio of peak amplitudes of mitoGCaMP6m and Fura-2 ΔF/F transients elicited in dendrites by suprathreshold synaptic stimuli and current pulses Data obtained from 8 to individual neurons. It is well that the of Ca2+ entry into the spines is via the et al., and which require glutamate and depolarization to the (Nowak et al., 1984). of by bath µM) almost completely and the mitoGCaMP6m transients evoked by the paired APs in the dendrites (Figure supplement 2). Discussion electrical cytosolic and mitochondrial Ca2+ imaging in L5 pyramidal neurons, we found that relatively spike the firing with cortical information in and produces rapidly decaying mitochondrial Ca2+ transients in all neuronal compartments. In contrast to a report of of mitochondrial Ca2+ transients to neuronal activity and cytosolic Ca2+ transients in in cortical neurons et al., 2019), data a relationship between neuronal electrical activity, cytosolic and mitochondrial Ca2+ studies (Ashrafi et al., 2020; et al., et al., 2016; et al., have revealed the long mitochondrial Ca2+ transients in presynaptic terminals of cultured central neurons. While this extremely rate of Ca2+ clearance reflect compartmental of mitochondrial Ca2+ handling at the presynaptic it seems more likely to be due to small mitochondrial Ca2+ signals in of cultured