Journal: Neural Regeneration Research

Article Title: MicroRNA-502-3p regulates GABAergic synapse function in hippocampal neurons

doi: 10.4103/NRR.NRR-D-23-01064

Figure Lengend Snippet: Patch-clamp analysis of cells treated with miR-502-3p. (A) Sweep plots showing the gamma-aminobutyric acid (GABA) current in the human-gamma-aminobutyric acid receptor A-α1/β2/γ2L (hGABAA-α1/β2/γ2L) human embryonic kidney (HEK) recombinant cells transfected with miR-502-3p agomiRs and miR-502-3p antagomiRs. The endogenous GABA channel was recorded at 300 μm GABA concentration for 4 seconds using the IonFlux system. (B) Quantification of GABA current in the parental, scramble control, miR-502-3p agomiRs, and antagomiR treated cells. The GABA current was reduced in agomiR transfected cells, while it was significantly increased in the miR-502-3p antagomiR transfected cells compared with scramble control. Experiments were performed a minimum 8 times in each group. The P values < 0.05 were considered statistically significant. miR: MicroRNA.

Article Snippet: Cells were patched with an intracellular solution suitably prepared for GABA current recordings (provided by Fluxion Biosciences).

Techniques: Patch Clamp, Recombinant, Transfection, Concentration Assay

Journal: iScience

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

doi: 10.1016/j.isci.2022.105830

Figure Lengend Snippet: 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 .

Article Snippet: 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.

Techniques: Activation Assay, Activity Assay, Control, Comparison

Journal: iScience

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

doi: 10.1016/j.isci.2022.105830

Figure Lengend Snippet: 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 .

Article Snippet: 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.

Techniques: Comparison, Activation Assay

Journal: iScience

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

doi: 10.1016/j.isci.2022.105830

Figure Lengend Snippet: 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.

Article Snippet: 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.

Techniques: Inhibition, Injection, Saline, Control

Journal: iScience

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

doi: 10.1016/j.isci.2022.105830

Figure Lengend Snippet: Serotonergic control of ANLS and neuronal energy levels Serotonin increases astrocytic cAMP and Ca 2+ 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.

Article Snippet: 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.

Techniques: Control