Mitochondrial Ca2+ uptake by the MCU facilitates pyramidal neuron excitability and metabolism during action potential firing

Groten, Christopher J.; MacVicar, Brian A.

doi:10.1038/s42003-022-03848-1

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Published: 02 September 2022

Mitochondrial Ca²⁺ uptake by the MCU facilitates pyramidal neuron excitability and metabolism during action potential firing

Communications Biology volume 5, Article number: 900 (2022) Cite this article

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Abstract

Neuronal activation is fundamental to information processing by the brain and requires mitochondrial energy metabolism. Mitochondrial Ca²⁺ uptake by the mitochondrial Ca²⁺ uniporter (MCU) has long been implicated in the control of energy metabolism and intracellular Ca²⁺ signalling, but its importance to neuronal function in the brain remains unclear. Here, we used in situ electrophysiology and two-photon imaging of mitochondrial Ca²⁺, cytosolic Ca²⁺, and NAD(P)H to test the relevance of MCU activation to pyramidal neuron Ca²⁺ signalling and energy metabolism during action potential firing. We demonstrate that mitochondrial Ca²⁺ uptake by the MCU is tuned to enhanced firing rate and the strength of this relationship varied between neurons of discrete brain regions. MCU activation promoted electron transport chain activity and chemical reduction of NAD⁺ to NADH. Moreover, Ca²⁺ buffering by mitochondria attenuated cytosolic Ca²⁺ signals and thereby reduced the coupling between activity and the slow afterhyperpolarization, a ubiquitous regulator of excitability. Collectively, we demonstrate that the MCU is engaged by accelerated spike frequency to facilitate neuronal activity through simultaneous control of energy metabolism and excitability. As such, the MCU is situated to promote brain functions associated with high frequency signalling and may represent a target for controlling excessive neuronal activity.

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Introduction

The activation of neurons and neuronal networks is fundamental to brain function and is critically dependent on ATP production by mitochondria^1,2,3,4_. In addition, these organelles influence reactive oxygen species production, apoptotic cascades, metabolite generation, and Ca²⁺ signalling^5,6,7,8,9. Of particular interest to the relationship between neuronal activity and mitochondrial function is the uptake of cytosolic Ca²⁺ by the mitochondria. This process relies on the inner mitochondrial membrane potential and is mediated by the mitochondrial Ca²⁺ uniporter (MCU). The MCU is a Ca²⁺-selective ion channel that is normally closed, but opens in response to elevations of cytosolic Ca²⁺^10,11,12. The Ca²⁺-dependent activation of the MCU is mediated by the regulatory subunits MICU1 and MICU2, which associate with the channel^11,13. Physiological elevation of mitochondrial Ca²⁺ via the MCU can enhance oxidative phosphorylation by facilitating the activity of the ATP synthase and dehydrogenases in the tricarboxylic acid cycle (TCA)^{8,14,15,16,17}. The uptake of Ca²⁺ by the mitochondria can also shape the magnitude and spatio-temporal dynamics of cytosolic Ca²⁺ and thereby control Ca²⁺-dependent signalling^{9,18,19,20,21}. In synaptic terminals, these effects of the MCU control synaptic transmission and promote local ATP production^2,22,23,24. Aside from its physiological role, excessive mitochondrial Ca²⁺ uptake can disrupt organelle function and may contribute to neuronal death during acute excitotoxicity and progressive degenerative disorders, such as Alzheimer’s and Parkinson’s disease^{25,26,27,28,29}. This highlights the importance of examining the mechanisms which govern mitochondrial Ca²⁺ uptake and the associated implications for neuronal function in the brain.

MCU activation may have a critical role in governing information processing in the brain. Numerous studies have demonstrated functions for mitochondrial Ca²⁺ uptake in neurons. However, these experiments were often performed under pathological conditions and in cultured cells^{2,27,30,31,32,33}. Moreover, previous measurements of mitochondrial Ca²⁺ in brain tissue were performed using Ca²⁺-sensitive dyes which do not selectively localize to the mitochondria and can have adverse effects^32,34,35. Consequently, the functional relevance of mitochondrial Ca²⁺ uptake to neurons during information processing in the mammalian brain is unclear. Interestingly, it was recently shown that the MCU in excitatory neurons is required for sustaining fast neuronal network oscillations in rodent hippocampal tissue³⁶. Moreover, in vivo imaging with a genetically encoded mitochondrial Ca²⁺ sensor revealed that sensory or motor evoked brain activation triggered robust mitochondrial Ca²⁺ elevations in the somatodendritic compartment of excitatory pyramidal neurons³⁷. These studies point to a potentially important role for the MCU and mitochondrial Ca²⁺ signalling during heightened brain activity, but several outstanding issues remain unaddressed. For example, the relationship between single neuron activity and mitochondrial Ca²⁺ uptake has not been established. This may be important considering that signalling pattern determines metabolic demand, synaptic transmission and plasticity^1,38,39,40. Additionally, discrete cell types and brain regions can exhibit distinct metabolic profiles, Ca²⁺ channels, and mitochondrial properties^41,42,43,44, indicating that the relevance of mitochondrial Ca²⁺ uptake to specific neuronal populations may vary. Lastly, the functional importance of mitochondrial Ca²⁺ uptake in the somatodendritic compartment of excitatory neurons during brain activity is not certain, but it could influence numerous processes, including adaptive energy metabolism, excitability, synaptic plasticity, and gene expression^21,45,46,47.

In the present study, we examined the functional relevance of the MCU during neuronal activation by determining the relationship between action potential firing and mitochondrial Ca²⁺ uptake in excitatory neurons of the cortex and hippocampus. Using a genetically encoded mitochondrial Ca²⁺ sensor, we demonstrate that the MCU activates in response to increasing action potential firing rate to initiate long-lasting mitochondrial Ca²⁺ elevations in the somatodendritic compartment of pyramidal neurons. By doing so, the MCU governed activity-dependent changes in mitochondrial energy metabolism and reduced the activation of the slow afterhyperpolarization (sAHP), which mediates negative feedback control of excitability. These results suggest that the MCU is engaged by changes in spike firing frequency to facilitate signal processing in pyramidal neurons of the brain.

Results

In situ characterization of cytosolic and mitochondrial Ca²⁺ in pyramidal neurons during action potential firing

In order to measure mitochondrial Ca²⁺ in pyramidal neurons, we drove the expression of mitoRGECO1.0 (mRGECO) in the rat brain by intracortical injection of adenoassociated virus (AAV) (Supplementary Fig. 1)^48,49. This sensor is a genetically encoded Ca²⁺-sensitive fluorescent protein that localizes to the mitochondria due to the presence of the targeting sequence from cytochrome c oxidase subunit VIII⁴⁸. mRGECO expression was controlled by the synapsin promoter to achieve its neuronal localization. This sensor was chosen because it is sensitive to modest Ca²⁺ concentrations (K_d = 0.48 µM) and has red emission, which allows for it to be imaged simultaneously with green fluorescent sensors⁴⁸. Following AAV injections, two-photon imaging of acute cortical and hippocampal brain slices showed the mitochondrial localization of mRGECO in the neuropil and pyramidal neuron cell bodies (Supplementary Fig. 1). In unstimulated cortical brain slices, mRGECO fluorescence was largely stable in the soma and neuropil over time (Fig. 1a, b). On occasion, there were local transient increases in mRGECO fluorescence (peak ΔF/F_o = 0.18 ± 0.026, n = 4 slices, N = 3) which presumably occurred as a result of spontaneous neuronal signalling (Fig. 1a, b). To monitor mitochondrial Ca²⁺ dynamics in relation to evoked neuronal activity, we performed whole-cell patch-clamp recordings from layer 3-5 pyramidal neurons expressing mRGECO. Cells were filled with Alexa-488 and confirmed to be pyramidal neurons based on the presence of a pyramid-shaped cell body, large apical dendrite, and multiple basal dendrites (Fig. 1c). Neuronal excitation was elicited using a 50 Hz, 4-sec train of depolarizing current pulses (+2 nA, 5 ms each), with each pulse consistently evoking a single action potential (Fig. 1c). Shortly after the onset of action potential firing, a punctate pattern of increased mRGECO fluorescence became apparent in both the soma and dendrites of pyramidal neurons, indicating that strong spike firing was sufficient to increase mitochondrial Ca²⁺ (Fig. 1c–e). The elevation of mitochondrial Ca²⁺ was very long-lasting and recovered to pre-stimulus baseline in a monoexponential fashion over several minutes (somatic τ = 145.5 ± 11.15 sec, n = 7, N = 4) (Fig. 1d). This protracted recovery of Ca²⁺ is consistent with known properties of mitochondrial Ca²⁺ dynamics and is mediated by the mitochondrial Na⁺/Ca²⁺ exchanger^48,50. Regional comparison of evoked responses showed that the peak mRGECO fluorescence change was greater in the soma relative to the apical dendrite (Fig. 1e). This may reflect differences in Ca²⁺ influx⁵¹ or the fraction of cell volume occupied by the mitochondria between the soma and dendrites.

**Fig. 1: Pyramidal neuron action potential firing triggers a long-lasting mitochondrial Ca²⁺ elevation mediated by the MCU.**

Ca²⁺ uptake into the mitochondria occurs primarily via the MCU, although other Ca²⁺ entry routes have been suggested^52,53,54,55. To examine this, we tested the effect of Ru360, an MCU inhibitor⁵⁶, on mitochondrial Ca²⁺ uptake during spike firing. As Ru360 has limited membrane permeability⁵⁷, it was dissolved in the internal solution of the pipette and delivered to neurons via intracellular dialysis (Fig. 1f). Ru360 at 20 µM, but not at 2 µM, reduced the magnitude of mRGECO fluorescence changes evoked by action potentials (Fig. 1f). Although a small residual mitochondrial Ca²⁺ elevation was usually present at this concentration of Ru360, these data confirm that the MCU is the principal route for mitochondrial Ca²⁺ uptake in pyramidal neurons during action potential firing.

Cytosolic Ca²⁺ derived from either Ca²⁺ entry across the plasma membrane or Ca²⁺ release from the endoplasmic reticulum can activate the MCU and increase mitochondrial Ca²⁺^9,10,58. We therefore, examined the relationship between cytosolic Ca²⁺ and mitochondrial Ca²⁺ levels during action potential firing. Removal of extracellular Ca²⁺ from the ACSF completely eliminated spike-evoked mitochondrial Ca²⁺ uptake (Fig. 2a), suggesting that the source of cytosolic Ca²⁺ driving the mitochondrial Ca²⁺ elevation is plasma membrane Ca²⁺ influx, rather than intracellular Ca²⁺ release. This Ca²⁺ influx is likely mediated by the activation of voltage-gated Ca²⁺ channels in the soma, as well as the dendrites through backpropagating action potentials^51,59,60. We next characterized the relationship between cytosolic and mitochondrial Ca²⁺ during action potential firing by simultaneously imaging mRGECO and cytosolic GCAMP6f (Fig. 2b, c), both of which have similar affinities for Ca²⁺ (GCAMP6f K_d = 375 nM; mRGECO K_d = 480 nM)^48,61. Delivering an action potential train (50 Hz, 4 sec) to pyramidal neurons co-expressing the two sensors caused an initial rapid increase in cytosolic Ca²⁺ in the somatodendritic compartment followed by a latent slower rise in mitochondrial Ca²⁺ (Fig. 2c–f; Supplementary Video 1). Following action potential firing, cytosolic Ca²⁺ levels returned to baseline within seconds, while mitochondrial Ca²⁺ recovered substantially more slowly, over minutes, to pre-stimulus baseline (Fig. 2g). Thus, transient increases in cytosolic Ca²⁺ may be capable of producing prolonged changes in mitochondrial function in the somatodendritic compartment that long outlast the period of action potential firing.

**Fig. 2: Simultaneous imaging of GCaMP6f and mRGECO reveals the distinct dynamics of cytosolic and mitochondrial Ca²⁺ during action potential firing.**

MCU-dependent mitochondrial Ca²⁺ uptake is tuned to action potential firing frequency

In vivo, pyramidal neurons in the cortex and hippocampus typically have low spike firing rates (<5 Hz) but can enter states of high-frequency action potential firing (10-50 Hz) during sensory processing or other cognitive tasks^39,62,63,64. Therefore, we next assessed the relationship between spike frequency and mitochondrial Ca²⁺ uptake to determine the relevance of the MCU to discrete firing intensities. mRGECO expressing cortical pyramidal neurons were patch-clamped and action potential trains were evoked at progressively increasing frequencies (1, 5, 10, 20, and 50 Hz). During low-frequency action potential trains (1 & 5 Hz), no apparent changes in the somatic mitochondrial Ca²⁺ signals were detected throughout the duration of stimulus (Fig. 3a, b). In contrast, the same number of action potentials applied at 10 Hz or greater resulted in rapid and large increases in mitochondrial Ca²⁺ (Fig. 3a, b). These responses became progressively larger and more prolonged with higher action potential firing frequency (Fig. 3a, b). The relationship between the peak mitochondrial Ca²⁺ response and spike frequency also appeared to plateau during high-frequency activity. This suggests that mitochondrial Ca²⁺ levels become constrained or that mREGCO saturates at the greater Ca²⁺ concentrations reached during high-frequency stimulation. To test the latter possibility, we repeated these experiments using the low-affinity mitochondrial Ca²⁺ sensor, mitoLaRGECO1.2 (mLaRGECO) (K_d = 12 µM)⁴⁸. Action potential evoked mLaRGECO fluorescence changes were smaller and briefer in duration relative to those measured with the high-affinity mRGECO sensor (Fig. 3b). Moreover, the spike frequency-response relationship measured with mLaRGECO was non-sigmoidal and lacked a clear plateau at high-frequency stimulation. This indicates that Ca²⁺ saturation of mRGECO likely contributed to the relationship between spike frequency and response magnitude (Fig. 3b). Collectively, these results demonstrate that MCU activation and subsequent mitochondrial Ca²⁺ uptake are highly sensitive to accelerated spike firing frequency in cortical pyramidal neurons.

**Fig. 3: The coupling between activity and mitochondrial Ca²⁺ loading in pyramidal neurons is dependent on action potential firing frequency and varies between brain regions.**

The coupling between neuronal activity and mitochondrial Ca²⁺ uptake varies between brain regions

Anatomically and functionally discrete brain regions can exhibit distinct neuroenergetic coupling, Ca²⁺ channel subtypes, and suite of mitochondrial proteins^41,42,43. Therefore, it may be possible for the relationship between neuronal activity and mitochondrial Ca²⁺ uptake to differ between distinct cell populations. To address this, we patch clamped mRGECO expressing hippocampal pyramidal neurons and compared the evoked responses to those measured in cortical neurons (Fig. 3c). Similar to cortical neurons, action potential trains in these neurons produced progressively greater mitochondrial Ca²⁺ uptake with higher action potential firing frequencies (Fig. 3d). We also assessed whether mitochondrial Ca²⁺ uptake occurred in response to activity patterns that are relevant to hippocampal neuron activity in vivo. To do this, we imaged CA1 pyramidal neurons while triggering theta burst firing. This stimulus pattern mimics the high-frequency action potential bursts (>50 Hz) that occur at theta frequency (~5 Hz) in CA1 neurons during rodent exploratory behaviour and learning processes^62,63,65,66. When CA1 neurons were triggered to fire high-frequency bursts applied at theta frequency, somatodendritic mitochondrial Ca²⁺ increased within seconds after the stimulus onset and remained elevated above pre-stimulus baseline for an extended period of time after the train (peak ΔF/F_o: 1.19 ± 0.26; time (sec) to 50% recovery: 87.41 ± 20.64, n = 4, N = 3) (Fig. 3e). This evidence supports a role for mitochondrial Ca²⁺ uptake during behavioural tasks associated with high-frequency signalling and theta bursting in vivo. The relationship between activity and mitochondrial Ca²⁺ uptake revealed that the peak train-evoked mRGECO fluorescence responses in hippocampal neurons plateaued at high action potential frequencies, similar to cortical neurons (Fig. 3f). Interestingly, low-frequency spike firing was much more effective at eliciting mitochondrial Ca²⁺ uptake in hippocampal neurons relative to those in the cortex (Fig. 3f). Consistent with this, the time to recovery from peak mitochondrial Ca²⁺ was also longer at low firing frequency in pyramidal neurons of the hippocampus relative to the cortex (Fig. 3f). These differences demonstrate a brain region-dependent variation in the strength of coupling between action potential firing and mitochondrial Ca²⁺ uptake by the MCU.

Adaptation of mitochondrial energy metabolism in pyramidal neurons during high-frequency action potential firing is mediated by the MCU

Mitochondrial Ca²⁺ uptake can depolarize the mitochondrial membrane potential, facilitate the activity of the electron transport chain, and promote the Ca²⁺-dependent activation of several key enzymes of the tricarboxylic acid cycle^30,67,68,69. We therefore examined how MCU-dependent mitochondrial Ca²⁺ flux controlled action potential evoked metabolic responses in single pyramidal neurons using two-photon microscopy of intracellular nicotinamide adenine dinucleotide hydride (NADH) autofluorescence^70,71,72. NADH is a coenzyme that is generated from the reduction of non-fluorescent NAD⁺ by the mitochondrial TCA cycle or via glycolysis in the cytosol⁷³. NADH is oxidized to NAD⁺ at complex I of the electron transport chain in order to generate the proton gradient used to synthesize ATP⁷³ (Fig. 4a). NADH autofluorescence can thus be used to assess changes in neuronal metabolic function. The measured autofluorescence will be referred to as NAD(P)H to acknowledge the inability to discern NADH from NADPH autofluorescence, although the former is more abundant in brain tissue^73,74.

**Fig. 4: Measurement of action potential evoked NAD(P)H dynamics in single pyramidal neurons in situ.**

Two-photon excitation of acute cortical brain slices revealed NAD(P)H autofluorescence in neuronal cell bodies that was distributed in a punctate pattern, similar to mitochondria labelled with mRGECO (Fig. 4b). We next performed patch-clamp recordings of cortical pyramidal neurons and measured NAD(P)H autofluorescence during the range of firing frequencies used to study mitochondrial Ca²⁺ uptake in our prior experiments. At firing frequencies above 1 Hz, there was a notable dip in NAD(P)H autofluorescence which became more prominent at higher action potential firing frequency (Fig. 4c–e). This dip reflects the oxidation of mitochondrial NADH by the electron transport chain in response to neuronal activation^30,69,73. At low firing frequencies, the NAD(P)H autofluorescence returned to pre-stimulus baseline shortly after the end of the stimulus (Fig. 4c, e). Conversely, after high-frequency action potential firing (20 & 50 Hz), the transient dip in NAD(P)H autofluorescence was followed by a long-lasting overshoot that eventually recovered to baseline after a minute or more (Fig. 4c). This NAD(P)H overshoot is consistent with a prolonged chemical reduction of NAD⁺ to NADH, which can result from mitochondrial TCA cycle activity or glycolysis in the cytosol^75,76. As the low-frequency trains were longer than the high-frequency stimuli, it may be possible that evoked metabolic responses are more likely to be influenced by prolonged laser exposure. We therefore examined whether NAD(P)H transients triggered by a 50-Hz train were influenced by 5 min of pre-exposure to the excitation laser at the illumination power used for imaging. Conditioning cells with the laser had no effect on the train-evoked NAD(P)H decrease or the long-lasting overshoot phase, suggesting that laser illumination had no influence on our results (Fig. 4f).

To test the involvement of mitochondrial metabolism, we examined the sensitivity of evoked NAD(P)H transients to rotenone (10 µM), which prevents NADH oxidation by complex I of the electron transport chain⁷⁷. Wash in of rotenone caused an elevation of neuronal autofluorescence in unstimulated neurons, consistent with a disruption of NADH oxidation in the presence of ongoing TCA cycle activity (Fig. 5a)⁷⁸. Subsequent stimulation of neurons in the presence of rotenone largely prevented the stimulus-evoked NAD(P)H transients (Fig. 5a, b). This data supports the interpretation that the measured autofluorescence changes primarily reflect mitochondrial NAD(P)H dynamics.

**Fig. 5: The MCU governs NAD(P)H dynamics during high-frequency action potential firing in pyramidal neurons.**

The growth in the magnitude of evoked NAD(P)H transients with increasing firing frequency paralleled the previously measured occurrence of mitochondrial Ca²⁺ uptake. We next examined a role for mitochondrial Ca²⁺ uptake in generating these NAD(P)H signals by blocking the MCU with Ru360 dialysis. Under these conditions, applying a 50-Hz, 4-sec train produced a briefer train-evoked dip in NAD(P)H autofluorescence relative to controls (Fig. 5a, b). Moreover, the latent NAD(P)H overshoot phase was substantially depressed in the presence of Ru360. These data indicate that the MCU has a prominent role in modulating NADH oxidation and the reduction of NAD⁺ to NADH during high-frequency action potential firing.

Mitochondrial Ca²⁺ uptake buffers cytosolic Ca²⁺ and reduces the slow afterhyperpolarization duration during high-frequency action potential firing

The detected rise in mitochondrial Ca²⁺ during high-frequency activity implies that mitochondrial Ca²⁺ uptake may shape the magnitude and temporal dynamics of cytosolic Ca²⁺ in the somatodendritic compartment⁹. We therefore assessed the extent to which MCU-dependent Ca²⁺ uptake influenced the relationship between cortical pyramidal neuron action potential firing frequency and evoked cytosolic Ca²⁺. This was accomplished using simultaneous whole-cell current-clamp recordings and measurement of cytosolic Ca²⁺ with the low-affinity Ca²⁺-sensitive dye Fluo5N. In control conditions there was an increase in the Ca²⁺ rise magnitude with progressively greater action potential firing frequencies (Fig. 6a). The influence of mitochondrial Ca²⁺ buffering was tested by dialyzing neurons with Ru360 (20 µM) to inhibit the MCU. At a low action potential firing frequency of 5 Hz there was no apparent influence of Ru360 on evoked cytosolic Ca²⁺ dynamics relative to control conditions (Fig. 6a, c). However, at action potential firing frequencies of 10 Hz or greater, Ru360 increased the peak cytosolic Ca²⁺ magnitude and time for post-train Ca²⁺ recovery (Fig. 6a–c; Supplementary Video 2). The most parsimonious explanation for these effects of Ru360 on cytosolic Ca²⁺ signals is disruption of the MCU, as this drug does not affect other determinants of Ca²⁺ signalling, such as the Na⁺/Ca²⁺ exchanger or voltage-gated Ca²⁺ channels⁵⁶. Consistent with this, the effects of Ru360 on cytosolic Ca²⁺ selectively occurred at firing frequencies which were demonstrated to evoke mitochondrial Ca²⁺ uptake with mRGECO. Measurement of the membrane voltage revealed the progressive growth in the magnitude of the post-train slow afterhyperpolarization (sAHP) with greater cytosolic Ca²⁺ and higher action potential firing frequency (Fig. 6a, c). The sAHP is mediated by the Ca²⁺-dependent activation of a neuromodulator-sensitive voltage-independent K⁺ current⁴⁶. This current is present in cells of multiple brain regions, where it plays a critical role in shaping neuronal signalling by promoting negative feedback control of excitability⁴⁶. Interestingly, blocking mitochondrial Ca²⁺ uptake with Ru360 led to a prolonged sAHP relative to controls, selectively during high frequency action potential firing (Fig. 6a, c). The prolongation of the sAHP closely correlated with the enlarged magnitude and protracted temporal dynamics of cytosolic Ca²⁺ elevations in the presence of Ru360. The dramatic effect of Ru360 on cytosolic Ca²⁺ and the sAHP kinetics suggests that mitochondrial Ca²⁺ buffering strongly controls the activation of the sAHP current (I_sAHP), and therefore neuronal excitability.

**Fig. 6: Mitochondrial Ca²⁺ uptake clamps cytosolic Ca²⁺ magnitude and reduces the slow afterhyperpolarization duration elicited by high-frequency action potential firing.**

Mitochondrial Ca²⁺ buffering reduces the coupling between Ca²⁺ influx and activation of the sAHP current in pyramidal neurons

To examine the interplay between mitochondrial Ca²⁺ uptake and the sAHP, we next measured I_sAHP in hippocampal CA1 pyramidal neurons, as it is well characterized in these cells⁴⁶. Under whole-cell voltage-clamp, applying a series of progressively longer transient step depolarizations, to initiate voltage-gated Ca²⁺ influx, resulted in the serial enlargement of the I_sAHP (Fig. 7a, b). The I_sAHP had a linear relationship with membrane voltage and a reversal potential consistent with a K⁺ conductance (Fig. 7c, d). In pyramidal neurons in which the MCU was blocked by dialysis with Ru360, there was no appreciable difference in the I_sAHP magnitude evoked by brief step depolarizations (Fig. 7a, b). However, with progressively longer step depolarizations (>600 ms), the I_sAHP in Ru360 became larger than control conditions (Fig. 7a, b). Examining the I_sAHP current-voltage relationship revealed that the depolarization evoked K⁺ current was sustained for a longer time in neurons exposed to Ru360, relative to controls (Fig. 7c, d). These findings indicate that Ru360 substantially enhances the activation of the Ca²⁺-activated K⁺ current underlying the I_sAHP, likely because of the reduced Ca²⁺ buffering of cytosolic Ca²⁺ when the MCU is blocked. We tested this possibility by examining the effect of Ru360 on the I_sAHP when pyramidal neuron Ca²⁺ buffering was enhanced by intracellular dialysis of the Ca²⁺ chelator BAPTA (10 mM). Under these conditions, BAPTA effectively eliminated the depolarization-evoked I_sAHP and largely prevented the enhancement of the current by Ru360 (Fig. 7e, f). These results are consistent with mitochondrial Ca²⁺ buffering preventing the Ca²⁺-dependent activation of the I_sAHP in pyramidal neurons.

Discussion

Mitochondrial Ca²⁺ uptake mediated by the MCU has long been implicated as a regulator of energy metabolism and intracellular Ca²⁺ signalling in neurons. Despite this, the functional relevance of MCU activation to neuronal activity in the brain has remained unclear. Using in situ two-photon imaging and electrophysiology, we revealed that: (1) The degree of mitochondrial Ca²⁺ uptake is a function of spike firing frequency in pyramidal neurons; (2) Increasing spike frequencies coupled with greater MCU-dependent Ca²⁺ uptake led to adaptive mitochondrial energy metabolism; (3) The coupling between action potential firing and mitochondrial Ca²⁺ uptake varied between brain regions; (4) Ca²⁺ uptake by mitochondria provides a remarkable degree of cytoplasmic Ca²⁺ buffering in pyramidal neurons during high-frequency firing and thereby controlled sAHP magnitude. We propose that MCU activation facilitates pyramidal neuron signalling by enhancing energy metabolism and excitability during the brain processes associated with high-frequency action potential firing (Fig. 8).

**Fig. 8: A functional model for mitochondrial Ca²⁺ uptake during shifts to high-frequency action potential firing in pyramidal neurons.**

Using a genetically encoded mitochondrial Ca²⁺ sensor, our experiments revealed the relationship between pyramidal neuron firing frequency and mitochondrial Ca²⁺ uptake in situ. Elevations in mitochondrial Ca²⁺ were largely absent during low-frequency action potential firing, but rapidly became larger and more prolonged in response to high-frequency trains or physiologically relevant bursting patterns. This relationship was also described in another recent study on cortical pyramidal neurons in brain slices⁷⁹. As the in vitro K_d of mRGECO is similar (0.48 µM) to the resting mitochondrial Ca²⁺ concentration^48,80, the lack of responsiveness at low activity levels likely reflects a true absence of Ca²⁺ uptake rather than unresponsiveness of the sensor. The apparent threshold level of action potential firing frequency required for mitochondrial Ca²⁺ uptake in cortical pyramidal neurons is consistent with the established relationship between cytosolic Ca²⁺ and MCU activation. This relationship shows a threshold and positive cooperativity, properties which are controlled by the Ca²⁺-sensitive MICU1 and MICU2 proteins^11,43. The Ca²⁺ threshold of MCU activation likely also contributed to the observed temporal dissociation between the rise in cytosolic and mitochondrial Ca²⁺ levels during the onset of action potential firing. While the specific Ca²⁺ concentration required for MCU activation in pyramidal neurons was not determined in our study, it has been estimated to be ~1 µM in non-neuronal cells¹¹. However, this threshold is likely to be lower in neurons, due to the abundance of MICU3, which enhances MCU activation^2,43. Additionally, recent in vivo imaging of pyramidal neurons provided evidence that mitochondrial Ca²⁺ uptake evoked by brain activity is facilitated by CaMKII function, which is sensitive to Ca²⁺ signal frequency and duration^37,81. Thus, such factors may govern the relationship between spike firing frequency and MCU activation, in addition to the absolute magnitude of cytosolic Ca²⁺ elevations.

Using single-cell imaging of intracellular NAD(P)H and electrophysiology we found that overt changes in adaptive energy metabolism increased in relation to spike firing frequency and paralleled the uptake of mitochondrial Ca²⁺. Moreover, mitochondrial Ca²⁺ uptake by the MCU governed NAD(P)H transients during high-frequency signalling. The partial influence of the MCU on the activity evoked NAD(P)H dip is consistent with enhanced NADH oxidation by the electron transport chain as a result of Ca²⁺ entry dissipating the mitochondrial membrane potential^30,69,78. As NADH oxidation was not eliminated by MCU blockade, electron transport chain activity is likely also facilitated by ADP produced by activity-dependent ATP consumption⁶⁹. The MCU dependence of the latent NAD(P)H overshoot phase indicates that this channel is also critical for increasing the mitochondrial NADH/NAD⁺ ratio. This can occur by enhanced chemical reduction of NAD⁺ to NADH mediated by the Ca²⁺-dependent facilitation of TCA cycle dehydrogenases^8,14,15,16. Measurements of activity dependent changes in NAD(P)H autofluorescence have been extensively used to monitor adaptive energy metabolism in brain tissue, but the importance of the MCU to these processes was not certain^73,82,83. For example, evoked NAD(P)H transients in the hippocampus have been shown to either be independent of or only partially dependent on Ca²⁺ influx from the extracellular space^82,83. Therefore, the present work clarifies the importance of mitochondrial Ca²⁺ entry via the MCU to metabolic adaptation in activated pyramidal neurons in situ. Our findings in cortical neurons are consistent with recent work in the hippocampal dentate gyrus, which showed that MCU knockdown partially disrupted the evoked mitochondrial NAD(P)H dip and strongly attenuated the delayed NAD(P)H overshoot phase⁷⁵.

In cultured neurons, direct measurements of presynaptic ATP revealed that mitochondrial Ca²⁺ uptake was required to sustain ATP levels during activity, and thereby maintained synaptic vesicle endocytosis². An analogous process likely occurs in the somatodendritic compartment in situ, as the NADH overshoot promoted by the MCU would be expected to contribute to oxidative phosphorylation¹⁴. As we found that substantial mitochondrial Ca²⁺ uptake only occurs during enhanced action potential firing rate, the MCU appears to serve as a conditional regulator of energy metabolism during periods of intense energy demand. This mirrors findings in cardiac tissue, where mitochondrial Ca²⁺ uptake is required for producing sufficient ATP to sustain action potential firing selectively during fight-or-flight accelerations in heart rate⁸⁴. The functional consequence of MCU-dependent ATP production in the somatodendritic compartment is unclear. Enhanced mitochondrial metabolic activity could contribute to the production of ATP required for maintaining membrane ion gradients^1,85 or supporting activity-dependent plasticity in response to high-frequency signalling⁸⁶. Aside from controlling neuroenergetic adaptation, changes in mitochondrial Ca²⁺ evoked by high-frequency firing could also alter the production of reactive oxygen species or mitochondrial metabolites, which can serve as cellular signalling molecules^7,87.

Our results provided evidence that the degree of coupling between neuronal activity and mitochondrial Ca²⁺ uptake differs between discrete brain regions. The greater coupling between spike firing and MCU activation in hippocampal neurons relative to cortical neurons can be accounted for by various factors. One explanation is cell type-dependent variation in the properties of evoked cytosolic Ca²⁺ signals. For instance, discrete neuronal populations exhibit unique Ca²⁺ buffering capacities⁸⁸ and the relative abundance of voltage-gated Ca²⁺ channel types can also vary, with some channel classes showing differential efficacy in triggering Ca²⁺ uptake by the mitochondria^{21,88,89,90,91}. The differential coupling could also reflect regional differences in intrinsic mitochondrial properties, such as energy metabolism^42,44 or the function of the MCU. For example, although the MCU complex is ubiquitously expressed, emerging evidence suggests that mitochondrial Ca²⁺ uptake in non-neuronal cells may vary substantially as a result of post-translational modification^92,93 or changes in the relative expression of the MCU and the MICU regulatory subunits^{41,43,94,95,96}. Interestingly, an analogous process may occur within the nervous system, as the expression profile of the MCU complex components is different between neurons and astrocytes as well as hippocampal subregions^41,94. Our work provides evidence that is consistent with such variation in the function of the MCU complex between brain regions. Physiologically, the brain region-dependent variation in coupling between activity and mitochondrial Ca²⁺ uptake may represent a mechanism for cell-specific adaption of energy generation. This may be advantageous, as discrete neuronal populations can exhibit distinct energy demands during signalling⁹⁷. In addition to its physiological role, excessive mitochondrial Ca²⁺ uptake can also contribute to metabolic dysfunction, reactive oxygen species generation, and neuronal death^27,98. Therefore, heightened mitochondrial Ca²⁺ uptake in the hippocampal neurons could contribute to the sensitivity of this region to dysfunction in various pathologies, such as Alzheimer’s disease or ischemia⁹⁹, in which mitochondrial dysfunction and Ca²⁺ overload have been implicated^25,27.

Prior studies have demonstrated that Ca²⁺ buffering by the mitochondria clamps cytosolic Ca²⁺ concentration and thereby shapes aspects of synaptic transmitter release in central synapses^22,23,100. However, its functional relevance in other subcellular compartments of central neurons during signalling has not been well established^100,101. Our work demonstrates a potent effect of mitochondrial Ca²⁺ uptake on cytosolic Ca²⁺ levels in the somatodendritic compartment, selectively during high-frequency action potential firing. By doing so, mitochondrial Ca²⁺ uptake strongly reduced the cytosolic Ca²⁺ available to activate the K⁺ current underlying the sAHP. Aside from the canonical Ca²⁺-dependent sAHP, disruption of intracellular ATP production by MCU blockade could conceivably lead to the activation of a K_ATP channel-mediated sAHP¹⁰². Nevertheless, this seems unlikely to have contributed to our results considering that increasing intracellular Ca²⁺ buffering prevented the enhancement of the sAHP seen during MCU disruption. The interplay between mitochondrial Ca²⁺ transport, ion channel modulation, and excitability has been demonstrated in olfactory sensory neurons, myenteric neurons, invertebrate neurons, and non-neuronal cells^{103,104,105,106}. However, it has not been established whether this link between mitochondrial Ca²⁺ uptake and excitability also occurs in central neurons. Our results reveal that Ca²⁺ buffering by the mitochondria exerts control over neuronal excitability in the brain. By reducing the coupling between Ca²⁺ influx and the activation of the sAHP, mitochondrial Ca²⁺ uptake is poised to promote neuronal signalling by disrupting negative feedback control of spike firing and synaptic integration^46,107,108. Such changes in the coupling between spike firing and activation of the sAHP can have meaningful consequences for information processing, as seen in the hippocampus during age-related learning disruption^109,110.

Collectively, our in situ study demonstrates that mitochondrial Ca²⁺ uptake in pyramidal neurons is tuned to enhanced action potential firing rate and promotes neuronal activity through a dual role in metabolic regulation and excitability control (Fig. 8). This dual role of mitochondrial Ca²⁺ uptake in the somatodendritic compartment could shape action potential output as well as synaptic integration and plasticity in the dendrites, where mitochondrial Ca²⁺ uptake was recently shown to occur during coincident pre- and post-synaptic activity⁷⁹. We expect that MCU activation in pyramidal neurons influences sensorimotor processing and other cognitive functions, as in vivo recordings of cortical and hippocampal pyramidal neurons have shown firing intensities within the range of our experimental stimuli^{39,62,111,112}. For example, CA1 pyramidal neurons transition from states of minimal action potential firing (<2 Hz) to periods of high frequency action potential bursts (>50 Hz) occurring at theta frequency, and thereby act as place cells during exploratory behaviour^62,63. Also, in the visual cortex, pyramidal neurons have little spontaneous activity (<1 Hz), but fire at ~10-20 Hz in response to the application of a visual stimulus³⁹. Consistent with MCU activation during information processing in the brain, a recent in vivo study demonstrated that mitochondrial Ca²⁺ elevations occur in neurons of the motor and visual cortex in response to motor behaviour or visual stimulation, respectively³⁷. Our results suggest that MCU activation may be engaged during such brain activity to sustain neuronal signalling through its influence on energy metabolism and excitability. Consistent with this, preventing mitochondrial Ca²⁺ uptake by the MCU has been found to disrupt spike firing rate increases in cortical neurons during sensory stimulation¹¹³. Moreover, the activation of fast neuronal network oscillations, which are associated with a multitude of cognitive processes, require high mitochondrial performance, and are prevented by MCU knockdown in excitatory neurons^4,36. A similar property may also be important in fast-spiking GABAergic interneurons, as these cells exhibit comparatively higher frequency firing and more prolonged action potential firing¹¹⁴. As activation of the MCU is primarily engaged during high-frequency signalling, this pathway could also serve as a target for controlling excessive neuronal activity in various brain pathologies.

Methods

Animals

Sprague Dawley rats were used for this study and were purchased from Charles River Laboratories. Rat housing and experimental procedures were performed in accordance with Canadian Council on Animal Care (CCAC) regulations and protocols approved by the University of British Columbia Animal Care Committee. Animals were group housed, fed ad libitum, and kept on a 12/12 hr light/day cycle. Rats were 17-28 days old when experiments were conducted.

Brain slice preparation

Rats were anesthetized with isoflurane in an induction chamber and subsequently decapitated. The brains were then quickly removed and placed in ice-cold slicing solution containing the following (in mM): 120 N-methyl-D-glucamine, 2.5 KCl, 25 NaHCO₃, 1 CaCl₂, 7 MgCl₂, 1.25 NaH₂PO₄, 20 D-glucose, 2.4 sodium pyruvate, and 1.3 sodium L-ascorbate. The slicing solution was continuously bubbled with 95% O₂ and 5% CO₂. Brains were then cut with a vibratome (Leica VT1200S) into coronal or horizontal slices (400 µm thick) for cortical and hippocampal experiments, respectively. Brain slices were immediately transferred to a chamber containing artificial cerebral spinal fluid (aCSF), which was warmed to 32 °C and continuously oxygenated with 95% O₂ and 5% CO₂. aCSF contained (in mM): 126 NaCl, 2.5 KCl, 26 NaHCO₃, 2 CaCl₂, 1.5 MgCl₂, 1.25 NaH₂PO₄, and 10 D-glucose, pH 7.3–7.4, osmolarity 310 mOsm. A Ca²⁺-free ACSF solution was used to test the involvement of extracellular Ca²⁺ entry on mitochondrial Ca²⁺ signals, and contained (in mM): 126 NaCl, 2.5 KCl, 26 NaHCO₃, 3.5 MgCl₂, 1.25 NaH₂PO₄, 10 D-glucose, 1 EGTA.

AAV injections

For AAV injections, neonatal rats, 1-3 days old, were anesthetized with isoflurane and placed in a stereotaxic frame. A glass micropipette attached to a Hamilton Syringe was then used to pierce the skull unilaterally at a region ~2/3 the distance between lambda and the eye. AAV solution (2 uL total; titer: 1.0 × 10¹³-1.5 × 10¹³ genome copies/mL) was then injected into the cortex at a rate of 600 nL/min using a motorized stereotaxic injector (Stoelting). Following injections, animals recovered from anesthesia and were then returned to the home cage. After 2-4 weeks, animals were sacrificed to prepare acute brain slices for experiments. AAVs were purchased from the Canadian Neurophotonics Platform Viral Vector Core Facility and Addgene: AAV9-synapsin-GCAMP6f (Addgene, 100837-AAV9); AAV2/9-synapsin-mitoRGECO1.0 (Neurophotonics); AAV2/9-synapsin-mitoLaRGECO1.2 (Neurophotonics); AAV2/9-synapsin-mitoGFP (Neurophotonics).

Chemicals and reagents

Drugs and dyes used for experiments are listed in final concentration (in mM): 0.02 Ru360 (Sigma Aldrich, 557440), 0.01 rotenone (Sigma Aldrich, R8875), 0.05 Alexa 488 hydrazide (Thermo Fisher Scientific, A10436), 0.05 Alexa 594 hydrazide (Thermo Fisher Scientific, A10438), 0.4 Fluo5N pentapotassium salt (Thermo Fisher Scientific, F14203). All drugs or chemicals were dissolved into the internal solution of the patch pipette or the aCSF. DMSO was used as the vehicle for rotenone and was diluted to a maximal working concentration of 0.1%.

Two-photon imaging in acute brain slices

Live brain slice imaging was performed using a two-photon laser-scanning microscope (LSM 7MP, Zeiss) equipped with a Zeiss water immersion objective lens (Plan-Apochromat 20×/numerical aperture 1.0). Excitation light was provided by a mode-locked Ti:Sapphire laser (Chameleon Ultra II, Coherent), which was pulsed at 80 MHz (140 fs per pulse). The excitation wavelength for each experiment was adjusted according to the fluorescent probes being imaged: mitoGFP & mRGECO, 1000 nm; NAD(P)H, 750 nm; Fluo5N & Alexa 594, 800 nm; mRGECO & Alexa 488, 1040 nm; mLaRGECO & Alexa 488, 1040 nm; mRGECO and GCAMP6f, 1040 nm. Emitted light was first passed through a 700 nm shortpass IR filter and then split with a 560 nm long-pass dichroic mirror (Chroma). Emitted light was then detected by two separate LSM BiG GaAsP detectors after passing through the appropriate emission filters (Chroma): green filter (520/60 nm) for MitoGFP, GCAMP6f, Fluo5N, Alexa488; blue filter for NAD(P)H (460/50 nm), red filter (630/75 nm) for mRGECO, mLaRGECO, Alexa594. Images were acquired using 8-16 line averaging and were processed and stored with Zen software (Zeiss).

Patch-clamp electrophysiology

Following brain slice preparation and a 30-min recovery period, tissue was transferred to the microscope recording chamber and perfused with aCSF at 2–3 mL/min. aCSF was bubbled with 95% O₂/5% CO₂ and warmed to 32 °C with a stage heater (Luigs & Neumann). Pyramidal neurons in the cortex (layer 3-5) and hippocampus (CA1 and CA3) were identified with transmitted light optics and an IR-1000 (DAGE-MTI) camera mounted to the LSM 7MP microscope. All neurons used for experiments were located >50 µm below the slice surface and had a resting membrane potential ranging from −55 to −70 mV. Neurons were patch-clamped using glass pipettes pulled from borosilicate glass capillaries (World Precision Instruments) with a P-97 Flaming/Brown Micropipette Puller (Sutter Instrument). Patch pipettes had a resistance of 3-8 MΩ and contained an intracellular solution comprised of the following (in mM): 110 K-Gluconate; 3 KCl; 10 Na-Gluconate; 2 MgCl₂; 4 K₂-ATP; 0.5 Na-GTP; 10 HEPES; 0.138 CaCl₂; 0.4 EGTA; pH adjusted to 7.2 with KOH and osmolarity adjusted to 290 mOsm. For high Ca²⁺ buffering experiments, the internal solution contained (in mM): 70 K-Gluconate; 3 KCl; 10 Na-Gluconate; 2 MgCl₂; 4 K₂-ATP; 0.5 Na-GTP; 10 HEPES; 4.15 CaCl₂; 10 K₄-BAPTA; pH adjusted to 7.2 with KOH and osmolarity adjusted to 290 mOsm. The free Ca²⁺ concentration of both internal solutions was calculated with WEBMAXC Standard (UC Davis) to be between 70-170 nM. For some experiments, the internal solution was supplemented with fluorescent dyes or drugs at the following concentrations (in mM): 0.05 Alexa 594 hydrazide; 0.4 K₅-Fluo5N; 0.05 Alexa 488 hydrazide; 0.02 Ru360. For all patch-clamp recordings, cells were dialyzed for a minimum of 15 min after whole-cell breakthrough before starting experiments. Neurons were confirmed to be pyramidal cells by morphological inspection, revealed by Alexa488 or Alexa594 fluorescence. Only neurons that possessed a pyramid-shaped cell body, large apical dendrite, and multiple basal dendrites were used in the study. Neuronal membrane voltage and current were recorded and controlled using a MultiClamp 800b amplifier, Digidata 1440 A digitizer, and Clampex 10.2 software (Axon Instruments, Molecular Devices). During voltage-clamp recordings, capacitance transients were compensated, and series resistance (8-20 MΩ) corrected to >40%. During whole-cell current clamp recordings, membrane voltage was maintained between −60 and −65 mV and series resistance was compensated using the bridge balance. Voltage offsets were nulled prior to seal formation and the membrane potentials presented were adjusted to correct for the liquid junction potential. Current and membrane voltage signals were low pass filtered by a built-in Bessel filter at 1 kHz and 3 kHz, respectively and digitized at 10 kHz.

Data analysis

All image analysis and image processing was performed using ImageJ (v 1.53). Prior to analysis, images were first processed to correct for translational movement using the correct 3D drift plugin on ImageJ. Activity-evoked fluorescence changes for mRGECO, mLaRGECO, GCAMP6f, and NAD(P)H were measured from a somatic region of interest (ROI) in pyramidal neurons and were defined as ΔF/F_o = [(F₁) − (F_o)] / (F_o). Where F₁ and F₀ represent the fluorescence at a given time point and the mean baseline fluorescence, respectively. Background fluorescence subtraction was not performed for these experiments as all regions neighboring the ROI contained meaningful fluorescence signal. Changes in Fluo5N fluorescence were determined similarly, but using background fluorescence subtraction: ΔF/F_o = [(F₁ − B₁) − (F_o − B_o)] / (F_o – B_o). B₀ and B₁ represent the background fluorescence during the baseline period and at a given time point during the time series recording, respectively. The ROIs used for measuring cytosolic Ca²⁺ in the soma with Fluo5N were drawn to exclude the nuclear fluorescence signal. For some data sets, fluorescence changes were also measured from ROIs placed over the proximal apical dendrite. Spontaneous mRGECO fluorescence transients were quantified by measuring the number and magnitude of fluorescence responses from a grid of 10 ×10 µm ROIs mapped onto each experiment time series. A spontaneous event was defined as a fluorescence increase in a given ROI that was >3 standard deviations above the mean baseline fluorescence. For analysis, peak or min ΔF/F_o responses were defined as the maximal changes in fluorescence after the stimulus onset time. The Ca²⁺ rise time was defined as the time from stimulus onset to half of the peak train-evoked ΔF/F_o. The coupling ratio between mitochondrial Ca²⁺ and cytosolic Ca²⁺ was calculated as the ratio of mRGECO ΔF/F_o and GCAMP ΔF/F_o at 2-sec post-stimulus onset. The rate of Ca²⁺ recovery in the mitochondria or cytosol was quantified by fitting the recovery phase with a monoexponential decay function or by measuring the time span from the end of the stimulus to the point at which ΔF/F_o was 50% of the peak fluorescence response. Images presented for publication were minimally spatially filtered and in some cases were also averaged across multiple time points. For some images, a mask of the neuronal outline was produced by forming a binary image from the intracellular Alexa594 fluorescence signal.

The analysis of membrane voltage and the current was performed using Clampfit (v10.2). The sAHP duration was determined by measuring the time span between the end of the action potential train to the point of 50% recovery of the membrane voltage back to the pre-stimulus baseline. The I_sAHP magnitude was determined by quantifying the post-depolarization area above prestimulus baseline (ms*pA/pF). This value equates to charge and was measured starting at 30 ms after the end of the step depolarization.

Statistics and reproducibility

Statistics were performed using GraphPad Prism (v7). All summary data shown represent the mean ± standard error of mean. For each data set, the cell replicates and the animal replicates are described within the summary data and figure legends. The cell replicate number is represented by the ‘n’ value and the animal replicate number by the ‘N’ value. Unless otherwise stated, all presented summary data represent distinct samples. Outliers were identified using the ROUT test and removed prior to further statistical testing. The Shapiro-Wilk normality test was used to examine data sets for normality. If data were normal, a Student’s paired or unpaired t test was used to test for differences between two means, while a one-way ANOVA with a Dunnett’s multiple comparisons test was used to test for differences between multiple means and a control condition. If the data were not normally distributed, a Mann–Whitney U test was used for two means, whereas a Friedman test with Dunn’s multiple comparison test was used to compare multiple means to a control condition. A Welch’s t test was used to compare two data sets if the variances differed significantly. Tests were considered statistically significantly different at p ≤ 0.05. The exact p-values are indicated in the figure legends.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

Data availability

Source data used for summary figures are provided in the Supplementary Data 1 file. AAVs used in this study are available from the Canadian Neurophotonics Platform Viral Vector Core Facility and Addgene: AAV9-synapsin-GCAMP6f (Addgene, 100837-AAV9); AAV2/9-synapsin-mitoRGECO1.0 (Neurophotonics); AAV2/9-synapsin-mitoLaRGECO1.2 (Neurophotonics); AAV2/9-synapsin-mitoGFP (Neurophotonics). Raw data from the current study is available from the corresponding author on reasonable request.

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Acknowledgements

This work was supported by a grant from the Canadian Institutes of Health Research (FDN-148397 to B.A.M.). C.J.G was supported by postdoctoral fellowships from the Canadian Institutes of Health Research and the Michael Smith Foundation for Health Research. The authors would like to thank Drs. Nicholas Weilinger, Louis-Philippe Bernier, and Stefan Wendt for their insightful feedback on the manuscript draft. We would also like to thank Rayshad Gopaul and Lucy Yang for their excellent technical assistance.

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Djavad Mowafaghian Centre for Brain Health, University of British Columbia, Vancouver, V6T 1Z3, Canada
Christopher J. Groten & Brian A. MacVicar

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Christopher J. Groten
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C.J.G. and B.A.M. conceived and designed the study. C.J.G. performed all experiments and data analysis. C.J.G and B.A.M. wrote and edited the manuscript.

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Groten, C.J., MacVicar, B.A. Mitochondrial Ca²⁺ uptake by the MCU facilitates pyramidal neuron excitability and metabolism during action potential firing. Commun Biol 5, 900 (2022). https://doi.org/10.1038/s42003-022-03848-1

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Received: 07 February 2022
Accepted: 16 August 2022
Published: 02 September 2022
DOI: https://doi.org/10.1038/s42003-022-03848-1

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