Figure 6.

Dissociated Motor Neurons Are Spontaneously Active

(A) Representative immunocytochemistry of 1016A-derived neurons from dissociated motor neuron spheres dissociated and plated at day 15, cultured for 1 week, and stained with antibodies specific for MAP2 (green) and SYNAPSIN1 (red). Nuclei were counterstained with DAPI (blue). (A′) is an image captured at higher magnification of the boxed area in (A). Scale bar represents 20 μm.

(B) Representative immunocytochemistry of HUES8-derived motor neurons spheres seeded at day 15 on top of cultures of human myotubes. After 1 week the co-culture was stained with antibodies specific for SMI32 (green) and fluorescently labeled α-bungarotoxin (BTX) (red). Scale bar represents 20 μm. (B′) is an image captured at higher magnification of the boxed area in (B).

(C) Representative results from experiments in which HUES8-derived motor neurons were dissociated from spheres at day 15 and transduced with AAV Syn:GCaMP6s 3 days later. Time-lapse microscopy was employed to record fluctuations in fluorescence over time 1 and 3 weeks after transduction.

(D) Electrophysiological recordings of HUES8-derived neurons from motor spheres analyzed 12 weeks after dissociation. Left: representative traces showing spontaneous action potential firing. Right: representative traces showing evoked action potential firing upon current injection from 0 to 90 pA in 10 pA increments.

(E) Representative traces showing normalized fluorescence intensity of GCaMP6s after GABA and AMPA (both at 100 μM) application in HUES9-derived motor neurons cultured for 3 weeks after sphere dissociation.

See also Figure S6.