Differential acute impact of therapeutically effective and overdose concentrations of lithium on human neuronal single cell and network function

Abstract

Lithium salts are used as mood-balancing medication prescribed to patients suffering from neuropsychiatric disorders, such as bipolar disorder and major depressive disorder. Lithium salts cross the blood-brain barrier and reach the brain parenchyma within few hours after oral application, however, how lithium influences directly human neuronal function is unknown. We applied patch-clamp and microelectrode array technology on human induced pluripotent stem cell (iPSC)-derived cortical neurons acutely exposed to therapeutic (<1 mM) and overdose concentrations (>1 mM) of lithium chloride (LiCl) to assess how therapeutically effective and overdose concentrations of LiCl directly influence human neuronal electrophysiological function at the synapse, single-cell, and neuronal network level. We describe that human iPSC-cortical neurons exposed to lithium showed an increased neuronal activity under all tested concentrations. Furthermore, we reveal a lithium-induced, concentration-dependent, transition of regular synchronous neuronal network activity using therapeutically effective concentration (<1 mM LiCl) to epileptiform-like neuronal discharges using overdose concentration (>1 mM LiCl). The overdose concentration lithium-induced epileptiform-like activity was similar to the epileptiform-like activity caused by the GABA_A-receptor antagonist. Patch-clamp recordings reveal that lithium reduces action potential threshold at all concentrations, however, only overdose concentration causes increased frequency of spontaneous AMPA-receptor mediated transmission. By applying the AMPA-receptor antagonist and anti-epileptic drug Perampanel, we demonstrate that Perampanel suppresses lithium-induced epileptiform-like activity in human cortical neurons. We provide insights in how therapeutically effective and overdose concentration of lithium directly influences human neuronal function at synapse, a single neuron, and neuronal network levels. Furthermore, we provide evidence that Perampanel suppresses pathological neuronal discharges caused by overdose concentrations of lithium in human neurons.

Fig. 1. Concentration-dependent bi-modulatory impact of lithium chloride (LiCl) on human cortical networks recorded by MEAs.

A Schematic representation of the applied protocol to evaluate the cumulative dose–response to LiCl in the hiPSC-derived 3D-neural aggregate cultures with synchronous network activity (day 20–50 after seeding). B, i Representative examples of MEA-recordings (three individual channels are shown) with their corresponding B, ii Spike raster plot and population firing rate diagrams showing the properties of network activity in baseline and in response to therapeutic and overdose concentrations of LiCl. The black arrows are marking the population bursts (PB) and the red arrows with the red line mark the population super-bursts (PSB). C Diagrams illustrating the change of neuronal network parameters in response to increasing concentrations of LiCl in synchronously active human neuronal networks. D The properties of PSB activity in overdose concentrations of LiCl. The data are presented as mean ± standard deviation, n = 10, N = 3. One-way ANOVA with Dunnett’s correction (baseline compared to the indicated group) was applied to calculate the shown p values.

Fig. 2. Overdose concentration of LiCl-induced epileptiform activity is comparable to GABA_A receptor antagonist-induced epileptiform activity in human cortical networks.

A Schematic representation of the applied protocol to evaluate the chemically induced epileptiform activities with LiCl and picrotoxin (PTX) in the hiPSC-derived 3D-neural aggregate cultures with synchronous network activity (day 20–50 after seeding). B Representative MEA-recordings (three individual channels are shown) showing the properties of network activity at baseline and after the application of (B, i) 10 mM LiCl or (B, ii) 50 µM PTX. C Representative MEA-recordings (five-minute, one channel) showing the epileptiform activity induced by C, i 10 mM LiCl or C, ii 50 µM PTX. The blue lines are marking the different stages of epileptiform activities: the population super-bursts (PSB) are proceeded by prominent spiking and are followed by a period of complete inactivity or silence. C Spike raster plots and population firing rate diagrams showing the epileptiform events induced by C, iii LiCl and C, iv PTX. D, i Diagrams illustrating the neuronal network parameters in response to 10 mM LiCl and PTX. D, ii Diagrams showing no change in network properties in the corresponding control experiments with equimolar NaCl and DMSO. n = 6, N = 2. The data is presented as mean ± standard deviation. A two-tailed, paired or unpaired t test was applied to calculate the shown p values.

Fig. 3. Patch–clamp recordings reveal the concentration-dependent impact of lithium chloride (LiCl) on excitability and synaptic function of human cortical neurons.

A Schematic representation of the applied protocol to evaluate the effect of therapeutic and overdose concentrations of LiCl on the hiPSC-derived neurons. B Examples of evoked firing responses to a 300 ms current injection of 45 pA into a current-clamped neuron at baseline, exposed to 1 mM, 5 mM LiCl, and after wash-out. C Examples of phase plane plots, calculated as the first derivative of the membrane potential at the initial AP, in a train of at least two APs, against the membrane potential. The black line denotes the AP threshold (at 10 mV/ms). D Diagrams illustrate the membrane potential parameters in the different conditions with individual cells shown as scattered dots. n = 11 (n = 8 for wash-out), N = 3. E, i Pie charts show the percentage of cells with spontaneous EPSCs and spontaneous IPSCs. A total number of cells is given in the center. E, ii Average frequency and amplitude of spontaneous EPSCs and IPSCs in the different conditions with individual cells shown as scattered dots. The relative frequency of the spontaneous events compared to baseline is also shown. E, iii The ratio of the average frequency of spontaneous AMPA/GABA, n = 10, N = 3. The experiments were done in a paired setup. The data are presented as mean ± standard deviation. One-way ANOVA with Dunnett’s correction (baseline compared to the indicated group) was applied to calculate the shown p values. For comparing the “wash-out” group, a paired t test was used for p value calculation. In the diagrams showing relative values, the p values were calculated by unpaired t test.

Fig. 4. Perampanel suppresses the synchronous neuronal activity in concentration-dependent manner.

A Schematic representation of the applied protocol to evaluate the cumulative dose–response of hiPSC-derived highly synchronous neuronal networks (day 30 after seeding) to the AMPA-receptor inhibitor Perampanel. B, i Representative examples of MEA-recordings (three individual channels are shown) with their corresponding. B, ii Spike raster plot and population firing rate diagrams showing the suppression of synchronous population bursting by increasing concentrations of Perampanel. C, i Dose–response curves showing the suppression of network parameters (number of spikes, % of spikes organized in population bursts-PB and number of PB) with increasing concentrations of Perampanel. The data is shown as % of change compared to baseline and represents the mean ± standard error of the mean values. The IC₅₀ values are given, n = 7, N = 2. C, ii Dose–response curves showing the stable network activity parameters with the application of DMSO vehicle control, n = 7, N = 2.

Fig. 5. Perampanel counteracts LiCl-induced epileptiform activity in human cortical networks.

A Schematic representation of the applied protocol to evaluate the applicability of Perampanel to reverse LiCl-induced epileptiform activity in human iPSC-derived synchronously active cortical networks (day 20–50 after seeding). B, i Representative examples of MEA-recordings (three individual channels are shown) with their corresponding. B, ii Spike raster plot and population firing rate diagrams showing the suppression of LiCl-induced epileptiform population super-bursting by Perampanel. C, i The suppression of network parameters with increasing concentrations of Perampanel. The data are presented as mean ± standard deviation, n = 11, N = 2. One-way ANOVA with Tukey’s correction (comparison between the indicated groups) was applied to calculate the shown p values. C, ii Diagrams showing no change in network properties in the corresponding control experiments with equimolar NaCl and DMSO, n = 6, N = 2. D Cell-attached patch–clamp recording of human iPSC-derived cortical neuron, showing the increased spontaneous activity after the application of overdose concentration of LiCl (5 mM) and its suppression by Perampanel (2 µM). E Whole-cell patch–clamp traces showing the increased frequency of spontaneous EPSCs after the application of overdose concentration of LiCl (5 mM) and its suppression after the application of Perampanel (2 µM). The boxes mark regions of interest showed at higher magnification below. PB population burst, PSB population super burst.