AAV2-hSyn-ChR2(H134R)-YFP

The zona incerta (ZI) is a subthalamic structure implicated in locomotion, fear, and anxiety. Although evidence suggests an anatomical connection between the ZI and the inferior olive (IO) and pontine nuclei (PN), how the ZI modulates neuronal activity in these regions remains to be determined. The researchers first tested this by monitoring the responses of single neurons in the PN and IO to optogenetic activation of channelrhodopsin-expressing ZI axons in wild-type mice in an awake in vivo preparation. Stimulation with short single pulses and trains of stimulation at 20 Hz evoked rapid responses in most recorded cells in both the PN and IO. Furthermore, the excitatory responses of PN neurons were proportional to the strength of ZI activation. Next, synaptic transmission at ZI-IO synapses was investigated using in vitro electrophysiology. Optogenetic activation of ZI axons elicited strong excitatory postsynaptic responses in IO neurons that remained robust to repetitive stimulation at 20 Hz. Overall, these results demonstrate a functional connection between the ZI-PN and ZI-IO pathways.

First, the presence of ZI projections to PNs was confirmed by injecting an adeno-associated virus expressing the excitatory opsin channelrhodopsin (ChR2) and a yellow fluorescent protein (YFP) reporter (AAV2-hSyn-ChR2 (H134R)-eYFP) into the ZI of wild-type C57BL/6 mice. Histological results showed the presence of ZI axons expressing ChR2 in PNs. Then, optodes were used to record extracellular unit activity in PNs of awake, head-restrained mice while stimulating ZI axons expressing ChR2 with 1-ms pulses of blue light (Figure 1A). Activation of ChR2-expressing axons near the recording site rapidly altered the firing of 77% of the PN neurons examined (Figure 1B). Three response types were observed: excitation (Ex), excitation followed by inhibition (Ex + In), and inhibition (In) (Figures 1B and 1C). Unresponsive cells were categorized as "no response" (Figure 1C). The mean latency of excitation (including inhibition after the excitation peak) was 2.39 ± 0.2 ms (Figure 1D), while the mean latency of inhibition was 2.8 ± 0.49 ms (Figure 1E). Together, these results confirm that ZI fiber activation can rapidly elicit responses in PN neurons.

Figure 1. A rapid short-duration optogenetic activation of zona incerta axons reliably elicits responses in the pontine nuclei. (Bhuvanasundaram R, et al., 2024)