Serotonergic neurons control cortical neuronal intracellular energy dynamics by modulating astrocyte-neuron lactate shuttle - PubMed (original) (raw)

. 2023 Jan 5;26(1):105830.

doi: 10.1016/j.isci.2022.105830. eCollection 2023 Jan 20.

Shinobu Hirai 1, Soojin Kwon 1, Daisuke Ono 2 3, Fei Deng 4 5, Jinxia Wan 4 5, Momoka Miyazawa 1 6, Takashi Kojima 1, Haruo Okado 1, Akihiro Karashima 7, Yulong Li 4 5, Kenji F Tanaka 8, Makoto Honda 1

Affiliations

Serotonergic neurons control cortical neuronal intracellular energy dynamics by modulating astrocyte-neuron lactate shuttle

Akiyo Natsubori et al. iScience. 2023.

Abstract

The central serotonergic system has multiple roles in animal physiology and behavior, including sleep-wake control. However, its function in controlling brain energy metabolism according to the state of animals remains undetermined. Through in vivo monitoring of energy metabolites and signaling, we demonstrated that optogenetic activation of raphe serotonergic neurons increased cortical neuronal intracellular concentration of ATP, an indispensable cellular energy molecule, which was suppressed by inhibiting neuronal uptake of lactate derived from astrocytes. Raphe serotonergic neuronal activation induced cortical astrocytic Ca2+ and cAMP surges and increased extracellular lactate concentrations, suggesting the facilitation of lactate release from astrocytes. Furthermore, chemogenetic inhibition of raphe serotonergic neurons partly attenuated the increase in cortical neuronal intracellular ATP levels as arousal increased in mice. Serotonergic neuronal activation promoted an increase in cortical neuronal intracellular ATP levels, partly mediated by the facilitation of the astrocyte-neuron lactate shuttle, contributing to state-dependent optimization of neuronal intracellular energy levels.

Keywords: Biological sciences; Neuroscience; molecular neuroscience.

© 2022 The Author(s).

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Conflict of interest statement

The authors declare no competing interests.

Figures

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Graphical abstract

Figure 1

Figure 1

Response of cortical neuronal intracellular ATP signals to optogenetic activation of raphe serotonergic neurons (A) Schematic illustration of the fiber photometric recording of intracellular ATP levels in cortical pyramidal neurons under photostimulation of serotonergic neurons in the raphe. CTX, cortex; DRN, dorsal raphe nucleus. (B) Representative traces of cortical neuronal intracellular ATP signals (Thy1-ATeam), EEG, and EMG signals with vigilance states of animals under serotonergic photostimulation. One-second blue light illumination for the opening of ChR2(C128S) in serotonergic neurons was followed by 1-s yellow light illumination for the closing of ChR2(C128S), with a 30-s interval between illuminations. Vertical blue and yellow lines indicate the 1-s illumination of each light color. (C) Traces of averaged Thy1-ATeam signals with state probabilities, EEG power density spectrum, and EMG activity under serotonergic photostimulation during wake, NREM sleep, and REM sleep states, respectively. Traces of Thy1-ATeam signals represent mean ± SEM (n = 5 sessions from 5 mice). (D) Area under the curve (AUC) of Thy1-ATeam signal responses to the optogenetic activation of serotonergic neurons. ∗p< 0.05 versus Control (Ctrl); two-sided Wilcoxon signed-rank test (n = 5 sessions from 5 mice). In the control condition, 1-s yellow light illumination was used instead of blue light. (E) Comparison of the AUC, peak value, and the peak time of Thy1-ATeam signal responses to serotonergic activation across the states for the data in (C). ∗p< 0.05 versus Wake; Friedman test with post hoc Steel-Dwass test (n = 5 sessions from 5 mice). (F) Comparison of fluctuations in Thy1-ATeam signals under serotonergic photostimulation-induced awakening and spontaneous awakening from NREM sleep (left) and REM sleep state (right), respectively (n = 5 sessions from 5 mice). (G) Comparison of the peak values and peak times of Thy1-ATeam signals under serotonergic photostimulation-induced and spontaneous awakening for the data in (F). ∗p< 0.05 versus Sponta; two-sided Wilcoxon signed-rank test (n = 5 sessions from 5 mice). (H) The effect of fluoxetine on the AUC of Thy1-ATeam signal responses to serotonergic photostimulation. ∗p< 0.05 versus Pre (before fluoxetine treatment); two-sided Wilcoxon signed-rank test (n = 5 sessions from 5 mice). (I) Effect of serotonin receptor subtype-selective antagonists on the AUC of Thy1-ATeam signal responses to serotonergic photostimulation. ∗p< 0.05 versus Pre (before each drug administration); two-sided Wilcoxon signed-rank test (n = 5 sessions from 5 mice). (J) The effect of selective noradrenergic lesions by DSP-4 (left) and muscarinic and nicotinic cholinergic receptor antagonists (scopolamine (Sco) and mecamylamine hydrochloride/methyllycaconitine citrate (M/M), respectively; right) on the AUC of Thy1-ATeam signal responses to serotonergic photostimulation. p< 0.05 versus Pre (before each drug administration); two-sided Wilcoxon signed-rank test (n = 5 sessions from 5 mice). Data are expressed as the mean ± SEM. See also Figures S1–S4.

Figure 2

Figure 2

Lactate mediates the increase in intracellular ATP levels in cortical neurons by activating raphe serotonergic neurons (A) Representative trace of diminished cortical neuronal intracellular ATP (Thy1-ATeam) signal responses to serotonergic photostimulation by topical administration of 4-CIN in comparison with Pre (before drug administration) in the same mouse. The blue line above the data represents the timing of serotonergic photostimulation for 30 s. (B) Effect of 4-CIN on cortical neuronal intracellular ATP dynamics under serotonergic photostimulation is shown as averaged traces of Thy1-ATeam signals before (Pre) and after the administration (n = 5 sessions from 5 mice). Vertical blue and yellow lines indicate the 1-s illumination of each light color. (C) Alteration of AUC and peak value of Thy1-ATeam signal responses to serotonergic activation by 4-CIN administration. ∗p< 0.05 versus Pre (before drug administration); two-sided Wilcoxon signed-rank test (n = 5 sessions from 5 mice). (D) Representative trace of blunted Thy1-ATeam signal responses to serotonergic photostimulation by topical administration of DAB in comparison with Pre (before drug administration) in the same mouse. (E) Effect of DAB on neuronal intracellular ATP dynamics under serotonergic photostimulation is shown as averaged traces of Thy1-ATeam signals (n = 5 sessions from 5 mice). (F) Alteration of AUC and peak value of Thy1-ATeam signal responses to serotonergic activation by treatment with DAB for the data in (D and E). ∗p< 0.05 versus Pre; two-sided Wilcoxon signed-rank test (n = 5 sessions from 5 mice). (G) Representative trace of sharpened Thy1-ATeam signal responses to serotonergic photostimulation by topical administration of L-lactate in comparison with Pre (before drug administration) in the same mouse. (H) Effect of L-lactate on neuronal intracellular ATP dynamics under serotonergic photostimulation. is shown as averaged traces of Thy1-ATeam signals (n = 5 sessions from 5 mice). (I) Alteration of AUC and peak value of Thy1-ATeam signal responses to serotonergic activation by treatment with L-lactate for the data in (G and H). ∗p< 0.05 versus Pre; two-sided Wilcoxon signed-rank test (n = 5 sessions from 5 mice). Data are expressed as the mean ± SEM. See also Figure S4.

Figure 3

Figure 3

Fluctuation of intracellular lactate levels of cortical neurons by activating raphe serotonergic neurons (A) Schematic illustration of the fiber photometric recording of intracellular lactate levels in cortical pyramidal neurons under the raphe serotonergic photostimulation in Tph2-ChR2(C128S) mice injected with AAV-CaMKII-Laconic. CTX, cortex; DRN, dorsal raphe nucleus. (B) Top, Laconic expression in CaMKII-positive pyramidal neurons in layer 5 of the motor cortex. Scale bar, 20 μm. Bottom, Percentage of neurons labeled with Laconic and CaMKII (1789 Laconic-positive cells and 1950 CaMKII-positive cells, in 4 mice). Lac, Laconic. (C) Traces of CaMKII-Laconic signals in the cortex under serotonergic photostimulation (Opt) and control light illuminations (Ctrl) during the NREM sleep state. Signal traces represent mean ± SEM (n = 5 sessions from 5 mice). Note that CaMKII-Laconic signal showed an initial trough (black arrow) followed by an increase (black arrowhead) under serotonergic photostimulation. Vertical blue and yellow lines indicate 1-s illumination of each light color. In the control condition, yellow light illumination was used in place of blue light. (D) Initial trough and following peak value of CaMKII-Laconic signal responses to serotonergic photostimulation. ∗p< 0.05 versus Control; two-sided Wilcoxon signed-rank test (n = 5 sessions from 5 mice). (E) Initial trough and following peak time of CaMKII-Laconic signals, compared with the peak time of Thy1-ATeam signals. ∗p< 0.05 versus peak time of Thy1-ATeam; Kruskal–Wallis test with post hoc Steel test (n = 5 sessions from 5 mice for CaMKII-Laconic and Thy1-ATeam, respectively). (F) Representative trace of altered CaMKII-Laconic signal responses to serotonergic photostimulation by topical administration of 4-CIN in comparison with Pre (before drug administration) in the same mouse. The blue line above the data represents the timing of serotonergic photostimulation for 30 s. (G) Comparison of averaged CaMKII-Laconic signal responses to serotonergic photostimulation between before and after the 4-CIN treatment (n = 5 sessions from 5 mice). (H) The effect of 4-CIN on trough and peak value of CaMKII-Laconic signal responses to serotonergic photostimulation. p< 0.05 versus Pre; two-sided Wilcoxon signed-rank test (n = 5 sessions from 5 mice). Data are expressed as mean ± SEM. See also Figure S5.

Figure 4

Figure 4

Cortical extracellular lactate concentration was raised by the activation of raphe serotonergic neurons (A) Schematic illustration of recording of cortical extracellular lactate concentrations under the raphe serotonergic photostimulation in Tph2-ChR2(C128S) mice. CTX, cortex; DRN, dorsal raphe nucleus. (B) Representative traces of cortical extracellular lactate concentrations and EEG and EMG signals under serotonergic photostimulation and control light illuminations. Vertical blue and yellow lines indicate 1-s illumination of each light color. In the control condition, yellow light illumination was used in place of blue light. (C) Traces of extracellular lactate signals in the cortex under serotonergic photostimulation (opt) and control light illuminations (Ctrl). Signal traces represent mean ± SEM (n = 5 sessions from 5 mice). We refer to the early phase during serotonergic photostimulation for 30 s and the late phase after the photostimulation, respectively. (D) AUC in the early phase (left) and late phase (middle), and peak value of late phase (right) of the extracellular lactate response to serotonergic photostimulation for the data in (C). ∗p< 0.05 versus Control; two-sided Wilcoxon signed-rank test (0–30 s for early phase and 30–400 s for late phase; n = 5 sessions from 5 mice). (E) Traces of averaged cortical extracellular lactate dynamics under serotonergic photostimulation-induced awakening (Opt) and spontaneous awakening (Sponta) from NREM sleep (n = 5 sessions from 5 mice). (F) Comparison of AUC in early phase (left) and late phase (middle), and peak value (right) and its time (below right) of the extracellular lactate dynamics between serotonergic photostimulation-induced and spontaneous awakening for the data in (E). ∗p< 0.05 versus Sponta; two-sided Wilcoxon signed-rank test (n = 5 sessions from 5 mice). (G) Comparison of averaged cortical extracellular lactate dynamics responding to serotonergic photostimulations between before (Pre) and after the fluoxetine treatment. Note that a temporary dip (black arrow) appeared immediately after the peak in the early phase under the fluoxetine treatment, so they were subsequently analyzed separately as early positive and negative phases. (H) The effect of fluoxetine on the AUC of the early positive phase (left), early negative phase (middle), and late positive phase (right) of the extracellular lactate response between before (Pre) and after the treatment for the data in (G). ∗p< 0.05 versus Pre; two-sided Wilcoxon signed-rank test (0–30 s for early phase and 30–400 s for late phase; n = 5 sessions from 5 mice). (I) The effect of fluoxetine on peak value and the peak time of the extracellular lactate response between before and after the treatment for the data in (G). ∗p< 0.05 versus Pre; two-sided Wilcoxon signed-rank test (n = 5 sessions from 5 mice). All traces and data of extracellular lactate signals represent as mean ± SEM.

Figure 5

Figure 5

Response of intracellular cAMP and Ca2+ signals in cortical astrocytes to the activation of raphe serotonergic neurons (A) Schematic illustration of the fiber photometric recording of astrocytic intracellular cAMP and Ca2+ signals in the cortex under the raphe serotonergic photostimulation in Tph2-ChR2(C128S) mice injected with AAV-GFAP-PinkFlamindo and AAV-GFAP-GCaMP6f, respectively. CTX, cortex; DRN, dorsal raphe nucleus. (B) Left, PinkFlamindo (top) and GCamp (bottom) expression in GFAP-positive astrocytes in layer 5 of the motor cortex. Scale bars, 20 μm. Right, for GFAP-PinkFlamindo (top), the percentage of cells labeled with PinkFlamindo and GFAP (2958 PinkFlamindo-positive cells and 3256 GFAP-positive cells, in three mice). For GFAP-GcaMP (bottom), the percentage of cells labeled with GCamp and GFAP (1394 GCamp-positive cells and 1507 GFAP-positive cells, in three mice). (C) Traces of GFAP-PinkFlamindo signals in the cortex under serotonergic photostimulation (Opt) and control light illumination (Ctrl). Signal traces represent mean ± SEM (n = 5 sessions from 5 mice). Vertical blue and yellow lines indicate 1-s illumination of each light color. In the control condition, yellow light illumination was used in place of blue light. (D) AUC of GFAP-PinkFlamindo signal responses to the raphe serotonergic photostimulation. ∗p< 0.05 versus Control; two-sided Wilcoxon signed-rank test (n = 5 sessions from 5 mice). (E) Traces of averaged control GFAP-mCherry fluorescence intensity fluctuations (left) and comparison between GFAP-mCherry and GFAP-PinkFlamindo fluorescent signals (right). Mann–Whitney test (n = 5 sessions from 4 mice for GFAP-mCherry and n = 5 sessions from 5 mice for GFAP-PinkFlamindo). (F) The effect of fluoxetine on AUC of GFAP-PinkFlamindo signal responses to serotonergic photostimulation. ∗p< 0.05 versus Pre (before drug administration); two-sided Wilcoxon signed-rank test (n = 5 sessions from 5 mice). (G) Traces of averaged GFAP-GCaMP signals in the cortex under serotonergic photostimulation (Opt) and control light illumination (Ctrl) (n = 5 sessions from 5 mice). (H) AUC of GFAP-GCaMP signal responses to serotonergic photostimulation. ∗p< 0.05 versus Control; two-sided Wilcoxon signed-rank test (n = 5 sessions from 5 mice). (I) Traces of averaged control GFAP-GFP fluorescence intensity fluctuations (left) and comparison between GFAP-GFP and GFAP-GCaMP fluorescent signals (right). ∗∗p< 0.01 versus GFAP-GFP; Mann–Whitney test (n = 5 sessions from 3 mice for GFAP-GFP and n = 5 sessions from 5 mice for GFAP-GCaMP). (J) The effect of fluoxetine, scopolamine, and DSP-4 on AUC of GFAP-GCaMP signal responses to serotonergic photostimulation. ∗p< 0.05 versus Pre (before the treatment); two-sided Wilcoxon signed-rank test (n = 5 sessions from 5 mice). (K) The effect of ACSF (control), TTX, SB204741, and SB242084 on AUC of GFAP-GCaMP signal responses to serotonergic photostimulation. ∗p< 0.05 versus Pre (before the administration); two-sided Wilcoxon signed-rank test (n = 5 sessions from 5 mice). Data are expressed as mean ± SEM. See also Figures S6 and S7.

Figure 6

Figure 6

Alteration of state-dependent cortical neuronal intracellular ATP signal dynamics by chemogenic inhibition of dorsal raphe serotonergic neurons (A) Schematic illustration of the fiber photometric recording of intracellular ATP levels in cortical pyramidal neurons in Sert-Cre::Thy1-ATeam mice injected with AAV-hSyn-DIO-hM4D(Gi)-mCherry into the dorsal raphe nucleus. CTX, cortex; DRN, dorsal raphe nucleus. (B) Representative traces of cortical neuronal intracellular ATP signals (Thy1-ATeam), EEG, and EMG signals with vigilance states of mice under saline or CNO administration. (C) Traces of averaged Thy1-ATeam signal in the cortex during the transition from NREM sleep to wake state under saline (control) or CNO administration (n = 6 sessions from 4 mice). (D) AUC (left), peak value (middle), and peak timing (right) of the Thy1-ATeam signal immediately after the transition from NREM sleep to wake state under saline or CNO administration. Two-sided Wilcoxon signed-rank test (n = 6 sessions from 4 mice). (E) Traces of averaged Thy1-ATeam signal during the transition from REM sleep to wake state under saline or CNO administration (n = 5 sessions from 3 mice). (F) AUC (left), peak value (middle), and peak timing (right) of the Thy1-ATeam signal immediately after the transition from REM sleep to wake state under saline or CNO administration. ∗p< 0.05 versus saline; two-sided Wilcoxon signed-rank test (n = 5 sessions from 3 mice). (G) Traces of averaged Thy1-ATeam signal during the transition from the quiet-awake to active-awake substate during the wake state under saline or CNO administration (n = 6 sessions from 4 mice). (H) AUC (left), peak value (middle), and peak timing (right) of the Thy1-ATeam signal immediately after the transition from the quiet-awake to active-awake substate under saline or CNO administration. Two-sided Wilcoxon signed-rank test (n = 6 sessions from 4 mice). (G) Traces of averaged Thy1-ATeam signal at the micro-awakening events during the NREM sleep under saline or CNO administration (n = 6 sessions from 4 mice). (J) AUC (left), peak value (middle), and peak timing (right) of the Thy1-ATeam signal at the micro-awakening events during the NREM sleep under saline or CNO administration. Two-sided Wilcoxon signed-rank test (n = 6 sessions from 4 mice). Bar graphs show mean ± SEM.

Figure 7

Figure 7

Serotonergic control of ANLS and neuronal energy levels Serotonin increases astrocytic cAMP and Ca2+ signals, triggering glycogenolysis for lactate production and its extracellular release. In turn, neurons increase intracellular ATP levels partly via lactate uptake from the extracellular space. CTX, cortex; DRN, dorsal raphe nucleus.

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