Figure 6. Optogenetic stimulations of transplanted human mGIN induce GABAergic postsynaptic responses in host hippocampal neurons.

(a) Left, a microscopic image showing both a GFP⁺ grafted human MGE cell and a GFP⁻ host pyramidal neuron in the CA3 of the hippocampus (dotted squares; cell bodies are indicated by arrows). These cells were labeled with biocytin-Streptavidin using recording pipettes (red). The grafted cell sends out projections towards the host pyramidal neuron. Right, microscopic images showing the soma and dendrites of the same GFP⁻ pyramidal neuron as in the left image. Projections from grafted human mGIN are shown in the middle panel (green, ChR2-GFP). (b) Top, blue light illumination evokes AP firings in GFP⁺ mGIN expressing ChR2 and induces the release of GABA at axon terminals, generating postsynaptic responses in the recorded GFP⁻ host neuron. Bottom left, blue light illumination (0.61 mW/mm², blue horizontal bar) did not induce ChR2-mediated current in GFP⁻ host neurons at −80 mV in voltage-clamp mode, indicating the lack of ChR2 expression. Bicuculline (30 µM) was added to inhibit GABAergic responses in the recorded host neuron. Bottom right, the comparison of ChR2-currents between grafted and host cells (n = 25 and 27 cells, respectively), which received the same blue light illumination (0.61 mW/mm²). *** p < 0.001. (c) Left, representative traces of postsynaptic currents recorded in a GFP⁻ host neuron. Postsynaptic responses were recorded at 0 mV in voltage-clamp mode and induced by photostimulations at 12.5 mW/mm² (1 ms duration, blue vertical line). Blue light illumination was applied every 10 seconds. Right, these postsynaptic currents were completely inhibited by the application of GABA_A receptor antagonist, bicuculline (30 µM) in the same neuron. (d) Left, 44 % of total 27 GFP⁻ host neurons displayed GABA_A receptor-mediated inhibitory postsynaptic currents (IPSC) induced by photostimulations. Middle, a summary graph showing the average amplitude of IPSCs before and after the application of bicuculline as in (c). ** p < 0.01. Right, a plot showing the average synaptic latency of IPSCs induced optogenetically and recorded in GFP⁻ host neurons (n = 10 neurons). The synaptic latency was quantified as the time interval between the start of photostimulations and the onset of synaptic responses. (e) Left, representative traces of postsynaptic currents recorded in a GFP⁻ host neuron. Postsynaptic currents were induced by blue light illuminations at 12.5 mW/mm² (1 ms duration, blue vertical line) and recorded in voltage-clamp mode at –80, −60, −40, −20, and 0 mV. Right, a current-voltage plot of the postsynaptic responses. Peak amplitudes of postsynaptic currents were plotted versus holding potential (V_h, closed circles). Linear regression (a dotted line) reveals the reversal potential of the postsynaptic currents (E_rev = −70 mV). The application of 30 µM bicuculline inhibited postsynaptic currents completely at all holding potentials examined (open circles). n = 3 neurons. (f) Overlaid traces of quantal IPSCs (qIPSC) indicate both successes (red traces) and failures (black traces). qIPSCs were induced by blue light illuminations (blue vertical line) and recorded in GFP⁻ host neurons as in (c). (g) Summary plots showing the average potency (quantal size) and release probability (P_r) of qIPSCs. n = 6 neurons. (h) A representative trace of IPSCs induced by train photostimulations. IPSCs were induced by blue light illumination applied at 1 Hz (12.5 mW/mm², 1 ms duration, blue vertical lines) and recorded in GFP⁻ host neurons at 0 mV in voltage-clamp mode. A trace on the right indicates the last evoked IPSC (a dotted circle). (i) A summary plot of IPSCs during 1 Hz train photostimulations as in (h). The peak amplitude of IPSCs was normalized to the first IPSC (a dotted line; n = 3). (j) Immunohistochemistry analysis of transplanted human mGIN. Hoechst (sky blue) was used as nuclear counter-stain. Arrows in magenta indicate GABAergic presynaptic terminals of GFP⁺ grafted cells (double-stained with ChR2-GFP and VGAT) and green arrows indicate inhibitory postsynaptic densities (stained with gephyrin, green) of GFPcells. (k–l) TEM images of grafted cells stained with DAB for human cytoplasm-specific antibodies (gray areas marked by asterisks). DAB⁻ host cells (no stain) receives synaptic inputs (arrows) from DAB⁺ grafted cells. Error bars are SEM.