Extended Data Fig. 6: ChR2-assisted circuit mapping and validation.
From: Thalamus-driven functional populations in frontal cortex support decision-making
a Left, calibration whole-cell recordings measuring vS1→vM1 connectivity using ChR2-assisted circuit mapping. Right, recordings from two example vM1 neurons during photostimulation of vS1 axons. The traces show 15 mins of recordings after TTX application. Green, membrane potential; blue, action potentials. TTX left EPSPs intact in the connected neuron (top, see light-induced EPSPs in the green trace after action potentials were eliminated). TTX abolished EPSPs in the unconnected neuron (bottom). b Pharmacology to verify synaptic connections. Left, photostimulation of vS1 axons elicits short-latency EPSPs in vM1 neurons. Application of TTX left EPSPs intact in a connected neuron (top). TTX abolished EPSPs in an unconnected neuron (bottom). Middle, data from a connected neuron. Application of TTX left EPSPs intact. Application of AMPAR antagonist NBQX and NMDAR antagonist AP5 abolished the remaining EPSPs, confirming that the remaining EPSPs resulted from synaptic depolarization. Thin lines, individual photostimulation repetitions; thick lines, mean. Right, data from all recordings tested with TTX, NBQX and AP5. Neurons tested with TTX, NBQX and AP5, n = 4; neurons tested with various TTX concentrations, n = 19. c Left, mean EPSP before and after TTX for all tested vM1 neurons. Right, EPSP latency of all vM1 neurons verified to be connected or unconnected using TTX. Mean ± SD across neurons (n = 28 neurons). Dots, individual neurons. Photostimulation power, 20 mW. Unconnected neurons with no EPSPs are shown on top. The EPSP latencies in vM1 neurons are overall faster than ALM neurons (Fig. 5f). Nevertheless, connected and unconnected neurons could be differentiated based on latency. d Left, connection probability of vS1 inputs in vM1 superficial (<570 µm) and deep layers (>570 µm). vS1 inputs preferentially excite vM1 superficial layers, consistent with24. Right, connection probability of M2 inputs to vM1 inputs. M2 inputs preferentially excite vM1 deep layers, consistent with24. e Limited anterograde infection of ALM neurons from virus injections in S1/S2, cALM, and ThalALM. Left, an example confocal image showing an ALM section 2 months after virus injection in S1/S2. Red, NeuN; green, ChR2 expression in S1/S2 input axons. Arrows indicate rare ALM neurons with ChR2 expression. Right, fraction of ALM neurons showing ChR2 or ReaChR expression for virus injections in S1/S2, cALM, and ThalALM (n = 3 mice each, 2–3 months after virus injection). The lack of ChR2 or ReaChR expression indicates that the light-induced EPSPs are due to ChR2 or ReaChR expressing long-range input axons. Scale bar, 10 µm.
