Serotonin altered mRNA expression in neurons and astrocytes

We measured the effect of serotonin on the expression of key genes involved in mitochondrial function and dynamics, neurotransmission and neuronal support, autophagy and cellular stress, and astrocyte-specific markers and functions (Fig. 1A–D) in healthy astrocytes after a 6-days serotonin treatment. Serotonin induced a downregulation of receptor 5-HT2B by 20% (Fig. 1B). SLC1A2, encoding the glutamate transporter EAAT2, was downregulated by 17% (Fig. 1D).

Fig. 1: mRNA expression in astrocytes and neurons.

mRNA expression of genes involved in A mitochondrial function and dynamics, B neurotransmission and neuronal support, C autophagy and cellular stress and D astrocyte-specific markers and function in serotonin-treated healthy astrocytes relative to untreated astrocytes. E mRNA expression of serotonin receptors 5-HT1A, 5-HT1A and 5-HT3A in untreated neurons relative to untreated astrocytes. mRNA expression of genes involved in F mitochondrial function and dynamics, G neurotransmission and brain function, and H autophagy and cellular stress in serotonin-treated healthy neurons relative to untreated neurons. All cells were derived from Ctl17. Ctl: untreated control. All data were analyzed using unpaired t-tests with Welch’s correction and presented as mean ± SEM. Significant differences were indicated with *p < 0.05, **p < 0.005.

We compared the expression of HTR1A, 2A and 3A in neurons and astrocytes. In neurons, the expression of HTR1A was 145-fold higher, mostly due to low expression in astrocytes with a Ct of 27 compared to 16 in neurons. While HTR2A showed a more modest 4-fold increase in neurons, the expression was robust in both astrocytes and neurons with Ct values of 19 and 14, respectively. HTR3A exhibited a 45-fold increase relative to astrocytes, due to low expression in astrocytes with a Ct of 24, compared to a Ct of 16 in neurons (Fig. 1E).

Then, we measured the expression of genes involved in mitochondrial function and dynamics, neurotransmission and brain function, and autophagy and cellular stress (Fig. 1F–H). Several genes involved in mitochondrial function and dynamics were downregulated. The expression of OPA1 and MFN1 involved in mitochondrial fusion dropped by 23% and 21%, respectively. DNM1L, coding for the mitochondrial fission protein DRP1, was downregulated by 21%. CYCS, coding for cytochrome C in the electron transport chain (ETC) was downregulated by 22%. VDAC1 and VDAC2, involved in metabolite transport across the mitochondrial outer membrane, were downregulated by 36% and 18%, respectively (Fig. 1F). Additionally, like in astrocytes, the expression of the gene encoding serotonin receptor 5-HT2B decreased by 47% (Fig. 1G). Regarding cellular stress response, the CAT gene encoding catalase was downregulated by 21% and the HIF-1α by 10% (Fig. 1H).

Astrocytic respiration and ATP levels were mildly altered by serotonin

We investigated the effect of a chronic serotonin treatment in astrocytes. We generated mature and functional astrocytes as demonstrated by the expression of the specific astrocytic markers GFAP, S100β, ALDH1L1 and EAAT1 (Fig. 2A).

Fig. 2: Effect of serotonin on the mitochondrial bioenergetics in astrocytes.

A Astrocytes markers. Immunofluorescence stainings show that cells express the typical mature astrocytes markers GFAP, ALDH1L1, S100β, and EAAT1. Scale bar indicates 20 µm. B Mitochondrial respiration. The oxygen consumption rate (OCR) was measured in untreated and serotonin-treated astrocytes following the Agilent XF Mito Stress Test protocol consisting of sequential injections of oligomycin (Oligo), carbonyl cyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP) and rotenone/antimycin A (Rot/AntA) to reveal different respiratory parameters. OCR values were normalized to 1000 cells by counting DAPI-stained nuclei. Experiments were conducted pairwise allowing direct comparison. Bar plots show normalized mean OCR values ± SEM. C ATP content was measured in untreated and serotonin-treated astrocytes using a luminescent assay and normalized to protein amount. Left: bar plot shows nM ATP per µg/mL proteins ± SEM. Right: before-after graph shows nM ATP per µg/mL proteins in individual paired replicates, i.e. astrocytes from the same passage, harvested and measured at the same time. D Mitochondrial membrane potential (MMP) was measured with the JC-1 dye and is indicated by the fluorescence ratio between JC-1 aggregates (fluorescing in red) over JC-1 monomers (fluorescing in green). Dot plot shows mean red/green ratios ±SEM. E Cytosolic calcium was measured as the Fura-2 fluorescence ratio F340/380 and is represented as mean ratio ±SEM (left). Mitochondrial calcium levels were measured using Rhod-2/AM and are presented as mean fluorescence intensity, in relative fluorescent unit ±SEM (right). F Cell size was analyzed by assessing area (pixels) of Fura-2/AM-loaded cells. Dot plot shows the number of pixels ±SEM. Ctl17 and Ctl18: healthy controls; Non-R: non-responder patient; Mito: mitochondriopathy patient; 5-HT: serotonin. Respiration data were analyzed a mixed-effect analysis one-way ANOVA. ATP content, MMP and calcium imaging data were analyzed with a one-way ANOVA without matching or pairing. Significant differences were indicated with *p < 0.05, **p < 0.005, ***p < 0.0005) and ****p < 0.0001.

An effect of serotonin on mitochondrial respiration has been reported in several cell types [26, 32]. Therefore, we measured the oxygen consumption rates (OCR) in key respiratory states in our astrocytes cell lines. We used the Agilent’s Mito Stress Test, which involves the serial application of specific inhibitors of ETC complexes.

In our model, serotonin did not show a strong influence on mitochondrial respiration, apart from a significant effect on Ctl17 astrocytes: basal respiration, proton leak, and non-mitochondrial respiration decreased. Serotonin treatment did not alter respiration in the Ctl18/Mito pair and the Mito patient’s astrocytes presented increased OCR in all mitochondrial respiratory states but proton leak (Fig. 2B).

To complement the respiratory measurements, we analyzed cellular ATP levels. Although the serotonin treatment did not lead to statistically significant changes overall, a trend was observed when pairing individual data points. Specifically, there appeared to be an overall increase in ATP content in Ctl17, Non-R and Mito astrocytes. Contrastingly, ATP levels in the Ctl18 astrocytes seemed to decrease upon serotonin exposure (Fig. 2C).

Serotonin influenced mitochondrial membrane potential and calcium homeostasis in astrocytes

The mitochondrial membrane potential (MMP) is an indirect measure of metabolic activity and capacity, reflecting the accumulation of protons in the intermembrane space. We assessed the MMP using the potential-dependent dye JC-1. Serotonin treatment increased the MMP of Non-R patient’s astrocytes, which effectively compensated the difference between Ctl17 and Non-R. In contrast, while serotonin increased the MMP in Ctl18, it markedly decreased it in Mito astrocytes resulting in a significantly lower MMP in Mito patient’s astrocytes (Fig. 2D).

Cellular Ca2+ homeostasis is crucial to mitochondrial function and plays an essential role in signaling, enzyme function, and metabolism. We investigated the effect of serotonin on astrocytic cytosolic and mitochondrial Ca2+ using Fura-2/AM and Rhod-2/AM, respectively. In Ctl17, serotonin increased cytosolic Ca2+ levels, whereas it decreased them in the Non-R patient. This effect reversed the difference between these two cell lines in untreated conditions. Similarly, serotonin treatment reversed the difference between Mito patient and Ctl18 astrocytes by decreasing cytosolic Ca2+ levels in the Mito patient’s astrocytes (Fig. 2E).

Furthermore, serotonin markedly increased mitochondrial Ca2+ levels in Ctl17 and Non-R astrocytes but decreased them in Mito astrocytes. While this increase was insufficient to normalize the significantly lower mitochondrial Ca2+ in Non-R, it did equalize the difference in mitochondrial Ca2+ levels between Ctl18 and Mito (Fig. 2E).

Serotonin did not alter cell size, measured in Fura-2/AM-loaded astrocytes. Mito patient’s astrocytes remained smaller than Ctl18 astrocytes (Fig. 2F).

In summary, serotonin significantly influenced Mito patient’s astrocytes by decreasing the MMP, cytosolic and mitochondrial Ca2+. In Non-R patient’s astrocytes, there was a consistent increase in MMP and mitochondrial Ca2+ levels after serotonin treatment. In Ctl17, serotonin increased both cytosolic and mitochondrial Ca2+, while it decreased the MMP in Ctl18.

Serotonin decreased Ca2+ and altered the MMP in neurons

Research suggest that serotonin is dysregulated in the brain of depressed patients [3]. It is also suggested to have a prenervous, trophic role on neurons, and to improve mitochondrial biogenesis and function [32]. To investigate this in our model, we generated mature cortical-like neurons using a method adapted from ref. [35]. The neurons expressed MAP2, β-III-tubulin, NeuN, PSD95 and VGLUT1, confirming their maturity and their identity [38,39,40,41].

As an indicator of mitochondrial function, we assessed the MMP in both somas and neurites using JC-1. In somatic mitochondria, serotonin increased MMP in both Ctl18 and Mito neurons. However, in neurites, serotonin treatment decreased the MMP in Non-R neurons, but increased it in Ctl18 neurons. As a result, the MMP was no longer higher in Mito neurons’ neurites compared to Ctl18 (Fig. 3B).

Fig. 3: Effect of serotonin on mitochondrial membrane potential (MMP), calcium homeostasis and dynamics in iPS-Neurons.

A Neuronal markers. Immunofluorescence stainings on untreated control neurons revealed that the induced neurons express typical neuronal cytoskeleton proteins MAP2 and βIII-Tubulin and neuronal nuclear marker NeuN. PSD95 and VGLUT1 expression indicated the presence of mature synaptic terminals. VGLUT1 expression suggested that most of the induced neurons are cortical-like glutamatergic neurons. Scale bars indicate 20 µm or 10 µm. B MMP was measured with the JC-1 dye and is indicated by the fluorescence ratio between JC-1 aggregates (fluorescing in red) over JC-1 monomers (fluorescing in green). Mitochondria from somas and neurites appeared on different focal planes and were therefore imaged separately. Representative images show red and green JC-1 fluorescence in the relevant structure. Scale bar indicates 20 µm. Dot plot shows mean red/green ratios ±SEM. C Calcium homeostasis in untreated and serotonin-treated neurons. Cytosolic calcium was measured as the Fura-2 fluorescence ratio F340/380 and is represented as mean ratio ±SEM. Mitochondrial calcium levels were measured using Rhod-2/AM and are presented as mean fluorescence intensity, in relative fluorescent unit ±SEM. D Cell size was analyzed by assessing area (pixels) of Fura-2/AM-loaded cells. Dot plot shows the number of pixels ±SEM. E Spontaneous calcium transients were analyzed in Fura-2/AM-loaded neurons. Example traces show representative calcium transients in a neuron (above) and a baseline subtracted calcium peak, illustrating maximal amplitude, rise time between 10 and 90% of maximal amplitude, the exponential fit used to calculate the time constant of decay Tau, and the full-width at half maximum (FWHM) (below). F Calcium transient dynamics in untreated and serotonin-treated neurons. Graphs show the maximum amplitude of the calcium transients (ratio 340 nm/380 nm ±SEM), the rise time, the time constant of decay Tau (s ± SEM) and the FWHM (s ± SEM). Ctl17 and Ctl18: healthy controls; Non-R: non-responder patient; Mito: mitochondriopathy patient; 5-HT: serotonin. All data were analyzed with a one-way ANOVA without matching or pairing. Significant differences were indicated with *p < 0.05, **p < 0.005, ***p < 0.0005 and ****p < 0.0001.

Ca2+ is an important second messenger involved in serotonin’s signaling through Gq protein-coupled receptors [42]. Moreover, Ca2+ homeostasis is crucial to mediate neurotransmission, and mitochondria play a pivotal role in balancing intracellular Ca2+ levels. Therefore, we investigated the effect of serotonin on Ca2+ homeostasis in neurons.

Serotonin consistently decreased both cytosolic and mitochondrial Ca2+ levels across all cell lines. For cytosolic Ca2+, this led to a normalization of differences between control and patient cells. For mitochondrial Ca2+, serotonin treatment resulted in higher levels in Non-R neurons compared to the matched Ctl17, but balanced the difference between Ctl18 and the Mito patient (Fig. 3C).

Serotonin decreased cell size in Ctl17 neurons but increased it in Ctl18 neurons, thereby eliminating the difference with Mito neurons (Fig. 3D).

Overall, serotonin markedly decreased Ca2+ levels in both the cytosol and mitochondria, while its impact on MMP varied. Notably, serotonin treatment often compensated for the differences between control and patient neurons, indicating an equalizing effect.

Serotonin altered the kinetics of Ca2+ transients

Besides measuring basal cytosolic Ca2+ levels, we recorded spontaneous Ca2+ transients over 20-minutes periods. We analyzed singles peaks to extract their amplitude, rise time, decay time and full width at half maximum (FWHM) (Fig. 3E).

The amplitude was increased by serotonin in Ctl17 and Mito neurons but decreased in Ctl18 neurons. This led to lower Ca2+ amplitudes in Non-R neurons and higher amplitudes in Mito neurons (Fig. 3F).

The rise time was markedly shortened by serotonin in Ctl17 neurons. Consequently, Non-R neurons exhibited a significantly longer rise time than Ctl17. The relative difference between Ctl18 and Mito remained similar before and after treatment, with longer rise time in Mito (Fig. 3F).

Serotonin prolonged the decay time in all cell lines but Ctl18, resulting in a lower rise time in Mito neurons, relative to Ctl18 (Fig. 3F).

The FWHM, measuring the duration of Ca2+ peaks, increased in Ctl18 upon serotonin treatment, normalizing the difference between Ctl18 and Mito (Fig. 3F).

In summary, serotonin treatment had varied effects on the kinetics of Ca2+ peaks. In Ctl17 neurons, serotonin made Ca2+ peaks higher, shortened rise time but prolonged decay time. In Non-R neurons, serotonin shortened decay time. In Ctl18 neurons, serotonin decreased amplitude but increased decay time and FWHM, resulting in lower and wider peaks. In Mito neurons, serotonin increased both the amplitude and the decay time of Ca2+ transients. Collectively, these findings demonstrate that serotonin had marked effects on spontaneous Ca2+ transients kinetics, although the effects varied among cell lines.

Serotonin depolarized RMP, decreased capacitance and increased current densities in neurons

Electrical activity is a hallmark of neuronal function. To investigate how serotonin influenced the biophysical properties of neurons, we performed whole-cell patch-clamp recordings. Moreover, 5HT2A signaling has been proposed to mediate serotonin’s effect on neuronal mitochondria [32]. Therefore, we additionally treated neurons with the specific 5HT2A antagonist M100907 (volinanserin) to test whether 5HT2A also mediated the effects of serotonin on electrophysiology.

Serotonin induced a significant depolarization of the resting membrane potential (RMP) in Ctl18 and Mito neurons. In Ctl17 and Non-R neurons, the RMP was also depolarized. This change was sufficient to normalize the difference between Non-R and Ctl17 neurons’ RMP. Remarkably, the depolarizing effect was lost when combining serotonin with M100907, indicating that it was mediated by 5-HTR2A signaling (Fig. 4A).

Fig. 4: Effect of serotonin and 5-HT2A antagonist M100907 on electrophysiological properties of iPS-Neurons.

A Resting membrane potential (RMP) in untreated and serotonin-treated neurons (left) and in DMSO-, serotonin+DMSO- and serotonin+M100907-treated neurons (right). Dot plots show mean RMP in mV ± SEM. B Membrane capacitance in untreated and serotonin-treated neurons (left) and in DMSO-, serotonin+DMSO- and serotonin+M100907-treated neurons (right). Dot plots show mean capacitance in pF ± SEM. C, D Sodium (INa) and potassium (IK) currents were recorded in voltage-clamp mode while holding the membrane potential at −80 mV (Vhold) and depolarizing in steps of 10 mV to provoke the opening of voltage-gated Na+ and K+ channels. Example traces show (C) the evoked Na+ and K+ current in untreated (black) and serotonin-treated (red) neurons, and (D) the resulting IV curves. E, F Sodium and potassium current densities at 0 mV in untreated and serotonin-treated neurons (left) and in DMSO-, serotonin+DMSO- and serotonin+M100907-treated neurons (right). Current measurements were normalized to the membrane capacitance to account for cell size variability (current density, pA/pF). E Dot plots show mean INa current density at 0 mV in pA/pF ± SEM and F mean IK current density at +20 mV in pA/pF ± SEM. Ctl17 and Ctl18: healthy controls; Non-R: non-responder patient; Mito: mitochondriopathy patient; 5-HT: serotonin. All data were analyzed with a one-way ANOVA without matching or pairing. Significant differences were indicated with *p < 0.05, **p < 0.005, ***p < 0.0005 and ****p < 0.0001.

Serotonin decreased membrane capacitance in Ctl17 and Ctl18. This equalized capacitance between control and patient neurons after treatment. Notably, we previously reported consistently smaller cell size in MDD and case study patients [33]. M100907 had no effect on capacitance (Fig. 4B).

We also measured active electrophysiological parameters, including sodium (Na+) and potassium (K+) currents. By holding the membrane potential at −80 mV and depolarizing it in steps of 10 mV, we induced the opening of voltage-gated Na+ and K+ channels (Fig. 4C, D). We normalized the currents to the soma size, reflected by membrane capacitance, resulting in current densities (pA/pF).

Remarkably, serotonin treatment consistently increased Na+ and K+ current densities in all cell lines. These changes did not alter the control/patient differences in Na+ currents. Contrastingly, the higher K+ currents observed in untreated Non-R and Mito neurons were equalized to the level of their controls. Interestingly, co-treatment with serotonin and M100907 led to a further increase in both Na+ and K+ current densities, however the underlying mechanism is not clear (Fig. 4E, F).

Serotonin altered the kinetics spontaneous postsynaptic currents and action potentials

Next, we held the membrane potential at −80 mV and recorded the current fluctuations corresponding to postsynaptic currents (PSCs) (Fig. 5A). We measured the decay time, rise time and amplitude of PSCs.

Fig. 5: Effect of serotonin on postsynaptic currents and action potentials kinetics.

A Spontaneous post-synaptic currents (PSCs) were recorded at a holding potential of −80 mV. Example traces show one single PSC (above) spontaneous PSCs (below). Graphs show B the time constant of decay Tau (ms ± SEM), C the rise time between 10% and 90% of the maximal amplitude, and D the maximum amplitude of the PSCs (pA ± SEM) in untreated and serotonin-treated neurons. E Spontaneous APs at −80 mV were analyzed individually. Example traces show a single AP trace illustrating threshold, amplitude and full width at half maximum (FWHM) (above) and spontaneous APs (below). Graphs show F mean threshold in mV ± SEM, G mean FWHM in ms ± SEM and H mean AP amplitude in mV ± SEM in untreated and serotonin-treated neurons. Ctl17 and Ctl18: healthy controls; Non-R: non-responder patient; Mito: mitochondriopathy patient; 5-HT: serotonin. All data were analyzed with a one-way ANOVA without matching or pairing. Significant differences were indicated with *p < 0.05, **p < 0.005, ***p < 0.0005 and ****p < 0.0001.

Serotonin had a marked influence on the kinetics of PSCs. Specifically, serotonin prolonged both the rise time and the decay time of the PSCs in all individuals but the Mito patient, where the effect was reversed. This resulted in prolonged PSCs in the Non-R and the Mito patient compared to their respective controls (Fig. 5B, C). On the other hand, serotonin increased the amplitude of PSCs in both Ctl18 and Mito neurons, maintaining the difference between them. Contrastingly, the amplitude decreased in serotonin-treated Non-R patient’s neurons, which made them smaller than in Ctl17 neurons (Fig. 5D).

We also investigated the effect of serotonin on spontaneous action potentials (APs). Therefore, we injected current to adjust the membrane potential to approximately −80 mV. We measured the threshold, FWHM and amplitude of the APs (Fig. 5E).

Serotonin decreased the AP threshold in all individuals but Ctl18. As a result, the difference between Ctl17 and Non-R was reversed, while Mito retained a lower threshold than Ctl18 (Fig. 5F). Serotonin reduced the width of the APs in Ctl17 neurons, which normalized its difference with Non-R neuron’s APs. In contrast, serotonin increased FWHM in Mito patient’s neurons, although insufficiently to equalize it to Ctl18 levels (Fig. 5G). Finally, APs amplitude increased in Ctl17 and Non-R neurons after serotonin treatment, which compensated the difference between them. Contrastingly, serotonin did not affect Ctl18 and Mito neurons’ APs (Fig. 5H).