Conclusion Summary: Optogenetic Interrogation of Cerebellar Circuitry of a Novel Mouse Model of Essential Tremor

Update as of July 1, 2019

Principal Investigator: Sheng-Han Kuo, MD,
Columbia University

We have made significant progress with the IETF grant to study the role of cerebellar circuitry in tremor in our novel mouse model with abnormal Purkinje cell (PC) synapses and essential tremor (ET)-like tremor (ho17J mouse model). Our plan is to selectively inhibit neuronal activities of PCs or inferior olivary nucleus (IO), and climbing fiber (CF)-PC synaptic transmission to assess the role of these neuronal activities in tremor.

Specific Aim 1: Identify the essential role of PC activities in tremor: We hypothesize that Purkinje cell activities are required for tremor in real time. Therefore, we will perform optogenetic inhibition of PC activities using NpHR (the light-driven ion pump halorhodopsin) and we will observe the effects on cerebellar oscillations and tremor in ho17J mice.

Progress: We performed experiments to inhibit PC activity using optogenetics and found that green light-activated NpHR (an inhibitory opsin) real-time inhibits tremor whereas the green light cessation causes instantaneous tremor rebound in ho17J mice. We repeated experiments in 5 mice, and all of them demonstrate at least 75% of tremor suppression during optogenetic-mediated PC inhibition. We also used control blue light, which does not activate NpHR, and blue light has no effects on tremor.

What we have learned: PC activity is a key element in the cerebellar circuitry for tremor.

SPECIFIC AIM 2: Identify the essential role of CF activities in tremor: We hypothesize that CF activities are required for tremor in real time. Therefore, we will perform pharmacological inhibition of CF activities by lidocaine microinfusion into the bilateral IOs and we will observe the effects on cerebellar oscillations and tremor in ho17J mice.

Progress: We performed lidocaine microinfusion experiments to suppress CF activities in ho17J mice. We also performed simultaneous IO activity monitoring. We found that lidocaine microinfusion into bilateral IOs causes slowing of IO firing frequency with simultaneous tremor frequency shift from 20Hz to 13Hz and more than 80% tremor amplitude suppression. We also found that the tremor suppressing effects last for about 30 minutes, and the tremor gradually returns. We repeated the experiments in 6 additional mice and all of them demonstrated consistent results.

What we have learned: CF activity is another key element within the cerebellar circuitry for tremor.

SPECIFIC AIM 3: Identify the essential role of CF-PC synaptic transmission in tremor: We hypothesize that CF-PC synaptic activities are required for tremor in real time. Therefore, we will use a novel optogenetic tool, mini-SOG, to achieve CF-PC synaptic specific inhibition, and we will observe the effects on cerebellar oscillations and tremor in ho17J mice.

Progress: We injected AAV8-miniSOG into bilateral IOs in ho17J mice, which results in robust expression of miniSOG at the CF terminals in the molecular layer, visualized by dual immunofluorescence of VGlut2 and calbindin. We then created a cranial window in ho17J mice with mini-SOG expression at the CF terminals and used blue light to scan the cerebellar surface to preferentially inhibit CF synapses close to the pial surface, which are presumed abnormal CF-PC synapses. This manipulation results in CF-PC synaptic inhibition for hours. We observed that CF-PC synaptic inhibition causes dramatic suppression of tremor in ho17J mice for approximately 2 hours and then tremor returns to baseline 6 hours later. Scanning with the control green light, which does not inhibit CF-PC synapses, does not suppress tremor.

What we have learned: CF-PC synaptic activity is the key element for tremor.