hESC-derived neurons adopt the bursting activity of a preexisting neural network. (A) Differential interference contrast and/or epifluorescent images of mouse cortical cultures (Left), hESC-derived neuronal cultures (Center), or mouse-human cocultures (Right) after 4 wk in vitro. (B) Representative traces of mouse (Upper) or human neuron (Lower) that received 500-ms voltage steps from −50 to +50 mV at 8 wk of coculture. (C) Representative current-clamp recordings from a mouse or human neurons at various time points. Bursting activity was routinely observed in mouse neurons (i) and human neurons between 4 (iii) and 8 wk (iv) of coculture, whereas no bursting was observed in hESC-derived cultures alone (ii). (D) Recordings in voltage-clamp display similar results as those in current-clamp but provide a uniform driving force (−70-mV holding potential) for quantification of the proportion of bursting cells. (E) Pooled data demonstrate a progressive increase in the proportion of hESC-derived neurons that display bursting at various times in coculture. Data are means ± SEM. (Scale bars, 100 μm.)

hESC-derived neurons display a reciprocal synaptic relationship with dissociated mouse cortical cultures. (A) Dual patch-clamp recordings from mouse (upper trace) and human (lower trace) that display repeatable, nearly simultaneous bursting in both cells when bursts were generated spontaneously by the culture. (B) Expanded time scale demonstrates that mouse activity preceded human activity (mean: 61.4 ± 9.5 ms). (C) Repeated 10-Hz light stimulations produced APs in hESC-derived neurons (Lower) and coincident bursting activity in mouse neurons (Upper). (D) Expanded time scale demonstrates that the first light stimulus preceded bursting by a mean duration of 73.7 ± 6.2 ms. (E) Dual patch-clamp recordings from a human (Upper) and mouse (Lower) neuron in which a spontaneous burst was triggered by the culture, followed by a light-induced burst generated by human neurons. (F) All spontaneous bursts and light-induced bursts (but not light-induced APs) were eliminated by application of CNQX (50 μM).

hESC-derived neurons act as presynaptic neurons within an existing neural network. (A) Traces from individual human (Upper) and mouse (Lower) neurons recorded in the presence of CNQX (50 μM) after coculture for 8 wk. Light stimulation of 1 Hz triggered escaped action currents in a hESC-derived neuron and unitary PSCs in a mouse neuron (middle trace), which could be blocked by application of the GABA_A receptor antagonist picrotoxin (50 μM; lower trace). (B) Expanded time scale illustrates the close temporal relationship among light stimulus, action current, and PSC. Note that traces from the human cell were essentially the same in the presence of CNQX alone or CNQX plus picrotoxin. (C) Representative traces from a mouse neuron recorded alone showing relatively long decay constants (τ = 20.2 ± 1.7 ms) and I-V relationship demonstrating a reversal potential of −41 ± 2.7 mV. (D) Representative traces from a mouse neuron recorded alone in the presence of AP5 (25 μM) showing relatively short decay constants (τ = 4.1 ± 0.7 ms) and I-V relationship demonstrating a reversal potential of 2.6 ± 0.7 mV. (E) Pooled data revealed a progressive increase in proportion of mouse neurons that displayed light-induced postsynaptic currents. Data are means ± SEM (*P < 0.05).

hESC-derived neurons regulate the excitability of host neurons following transplantation. (A) Fluorescent image of a mouse brain section in which transplanted ChR2-expressing hESC-derived neurons have been stained with human nuclear antigen and can be seen innervating various regions of the hippocampus. (B) Representative current-clamp recordings in a ChR2-mCherry⁺ neuron showing a single (Lower) and a 10-Hz train (Upper) of nonaccommodating APs in response to 70 pA of current injection. (C) Sequential voltage-clamp traces from a hESC-derived neuron that demonstrates the presence of spontaneous PSCs. (D) Current-clamp traces demonstrating the presence of light-induced APs at 5 Hz (Upper) and 10 Hz (Lower). (E) Fluorescent image of acute hippocampal slice containing ChR2-mCherry hESC-derived neurons (red) in close proximity to a recorded CA1 pyramidal neuron loaded with alexafluor-488 (green). (F) Voltage-clamp traces of CA1 pyramidal neuron demonstrating inward and outward currents (lower trace: expanded time scale for clarity of inward currents). (G) Current-clamp recordings of CA1 neuron showing a single (Lower) and a train (Upper) of accommodating APs in response to 70 pA of current injection. (H) Voltage-clamp recording of CA1 neuron that demonstrates the presence of repeatable light-induced PSCs (upper trace) that were blocked by picrotoxin (50 μΜ; lower trace). (I) Expanded time scale of currents in H illustrating the relatively long decay constants of LI-PSCs (τ = 20.2 ± 1.7 ms). (Scale bars, 100 μm.)