Article
Extended Data Fig. 6 | Electrophysiological properties of gradually
matured DA neurons. a, b, Schematic depiction of patch recording of
converted neurons in midbrain (a). According to this scheme, the f luorescent
dye Neurobiotin 488 (green) loaded in the electrode was used to mark cell
bodies in substantia nigra for patch clamp recording on brain slices. b, After
recording, the patched cells were confirmed to be RFP+TH+ to demonstrate the
recording being performed on newly converted neurons (scale bar, 20 μm).
Experiments were independently repeated 4 times with similar results.
c–e, Detection of spontaneous action potential (c) and relatively wider action
potential generated by newly converted neurons in comparison with
endogenous GABAergic neurons (d). e, Notably, hyperpolarization-activated
currents of HCN channels (Ih currents) were recorded at 12 weeks after, but not
6 weeks after, AAV-shPTB-induced neuronal conversion; these currents could
be specifically blocked with CsCl. The numbers of cells that showed the
recorded activity versus the total number of cells examined are indicated. Note
that the bottom trace is also shown in Fig. 2h. f, g, Extracellular recording
showing more converted neurons firing spontaneous action potentials at
12 weeks after transduction with A AV-shPTB than at 6 weeks after transduction.
The numbers of cells that showed the recorded activity versus the total number
of cells examined are indicated. g, The frequency of spontaneous spikes that
increased upon further maturation was further quantified. Data were based
on a total of 31 cells from 4 mice. Results show progressive maturation of
newly converted DA neurons in the brain. Statistical significance was
determined by two-sided Student’s t-test. h, Cortical neurons generated in
A AV-shPTB-transduced cortex, in contrast to a large population of RFP+TH+
cells in midbrain. As a control, A AV-shPTB was injected in cortex. After
12 weeks, RFP+ cells were co-stained with the cortical neuron marker CTIP2
(top) and CUX1 (bottom). Scale bars, 40 μm (main); 15 μm (magnified inset).
Note that RFP+ CUX1+ cells are rare in comparison to RFP+CTIP2+ cells,
indicative of different conversion efficiency in different layers of cortex.
Experiments were independently repeated twice with similar results.