Bringing to light the physiological and pathological firing patterns of human induced pluripotent stem cell-derived neurons using optical recordings

Abstract

Human induced pluripotent stem cells (hiPSCs) are a promising approach to study neurological and neuropsychiatric diseases. Most methods to record the activity of these cells have major drawbacks as they are invasive or they do not allow single cell resolution. Genetically encoded voltage indicators (GEVIs) open the path to high throughput visualization of undisturbed neuronal activity. However, conventional GEVIs perturb membrane integrity through inserting multiple copies of transmembrane domains into the plasma membrane. To circumvent large add-ons to the plasma membrane, we used a minimally invasive novel hybrid dark quencher GEVI to record the physiological and pathological firing patterns of hiPSCs-derived sensory neurons from patients with inherited erythromelalgia, a chronic pain condition associated with recurrent attacks of redness and swelling in the distal extremities. We observed considerable differences in action potential firing patterns between patient and control neurons that were previously overlooked with other recording methods. Our system also performed well in hiPSC-derived forebrain neurons where it detected spontaneous synchronous bursting behavior, thus opening the path to future applications in other cell types and disease models including Parkinson's disease, Alzheimer's disease, epilepsy, and schizophrenia, conditions associated with disturbances of neuronal activity and synchrony.

Keywords: GABA; action potential firing patterns; co-cultures; dark quencher genetically encoded voltage indicator; glutamate; iPSC-derived sensory neurons; inherited erythromelalgia; synchronous burst firing.

FIGURE 1

(A) In vitro cultivation and adeno-associated viruse (AAV) transduction paradigm for human induced pluripotent stem cell (hiPSC)-derived sensory neurons. RI, ROCK-Inhibitor, MMC: Mitomycin C. (B) Human induced pluripotent stem cell-derived sensory neuron (hiPSCdSN) express typical sensory neural marker, such as ISLET1, BRN3A, and PRPH, as well as peripheral sodium channels Nav1.7 and Nav1.8. (C) Dark quencher hybrid genetically encoded voltage indicator (dqGEVI) specifically labels the sensory neuronal cell membrane. EM, erythromelalgia.

FIGURE 2

Optical detection of spontaneous spiking in human induced pluripotent stem cell -derived sensory neurons (hiPSC–SNs) from an erythromelalgia (EM) patient and control. (A) Top: Images of two (Cell 1 in blue and Cell 2 in red) control hiPSC–SNs expressing dark quencher hybrid genetically encoded voltage indicator (dqGEVI). Somata were detected with an automated soma detection and evaluation procedure. Bottom: Raw imaging traces of cell 1 (blue) and cell 2 (red). (B) Pie-graphs showing the percentage of spontaneously firing cells from EM patient (Total N = 85, active N = 29) and control (Total N = 77, active N = 24). (C) Representative raw imaging traces of spontaneous firing behavior of hiPSC-SNs from the EM patient (bottom) and control (top). (D) Violin plots of the firing rate distribution in the EM patient human induced pluripotent stem cell-derived sensory neurons (hiPSCdSNs) (N = 45) and the control hiPSCdSNs (N = 27). The thin vertical black line indicates the 95% confidence interval. The thick vertical black line indicates the interquartile range. The median is indicated by the white dot. Spiking frequency is significantly higher in hiPSCdSNs from EM patients (Wilcoxon Rank test, WRT, p = 0.03).

FIGURE 3

Increased spontaneous bursting discharge in erythromelalgia (EM) patientdSNs. (A) Left: Representative raw imaging traces of control human induced pluripotent stem cell-derived sensory neurons (hiPSCdSNs) (top, orange) showing a sporadic firing pattern and EM patient hiPSCdSNs (bottom, green) showing a clear bursting pattern. Right: Log-binned Interevent interval (IEI) histograms of the example EM patient hiPSCdSNs trace exhibited two distributions that were well fit with multiple exponential distributions whereas control hiPSCdSNs were well fit with a single exponential. Yellow lines indicate exponential fits. Blue lines indicate the sum of the fits. (B) Comparison of the burstiness of EM patient hiPSCdSNs and control hiPSCdSNs. Filled black dots indicate the average values. Burstiness is significantly higher in EM patient hiPSCdSNs (EM patientdSNs N = 45, control hiPSCdSNs N = 27; WRT *p = 0.04). (C) Plot of the shifted IEIs for single cell examples of EM patient hiPSCdSNs and control hiPSCdSNs. Dotted black lines indicate clusters of short followed by short (S->S) Short followed by long (S->L) and L->S in the EM patient group. (D) Enlargement of the S->S cluster in (C) on a log scale.

FIGURE 4

Erythromelalgia (EM) patient human induced pluripotent stem cell–derived sensory neurons (hiPSC–SNs) are hypersensitive to mild increases in ambient temperature. (A) Representative raw imaging traces of control hiPSC-SNs (top, orange) and EM patient–SNs (bottom, green) at 37 and 40^°C. (B) Comparison of spike frequency filled black dots indicate average values. Error bars indicate SEM. [EM patientdSNs N = 10, control human induced pluripotent stem cell-derived sensory neurons (hiPSCdSNs) N = 6, Wilcoxon rank-sumtest, control: *p = 0.87, EM patient: *p = 0.027].

FIGURE 5

Optical detection of epileptogenic bursting behavior in human induced pluripotent stem cell (hiPSC)-derived forebrain neurons [induced forebrain glutamatergic neurons (iGluN) (80%) and GABAergic neurons (iGABAN) (20%)]. (A) Top: Automated detection of the somata of two example hiPSC-derived iGluN iGABAN neurons. (B) Cross-correlation between the fluorescence traces of two example neurons. (C) Upscaling of the dark quencher hybrid genetically encoded voltage indicator (dqGEVI) imaging system. Top: Automated detection of the somata of six hiPSC-derived forebrain neurons. Bottom: respective fluorescence traces.