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

Update as of January 28, 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 have spent the past 6 months to test NpHR expression in PCs in ho17J mice using AAV2-mediated approach. We have successfully achieved a good expression level of NpHR in PCs in ho17J motor cerebellum (lobule V and VI, responsible for cerebellar motor control). Importantly, we also achieved PC-specific expression of NpHR using PC-specific promoter, which allows to precisely inhibit PC activities. We now have a cohort of 10 ho17J mice with NpHR expression in PCs, and they also have optotrodes chronically implanted in the cerebellum for simultaneous light-activated inhibition and PC activity recording. It will take another 2 weeks (4-week post-surgery) to test how real time inhibition of PC activities can influence tremor.

Next steps: We will perform optogenetic inhibition of PC activities with simultaneous PC recording and tremor assessment in this cohort of ho17J mice in the next 2 months. We will spend another 1 month for data analysis for this aim.

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 have successfully inserted the microinfusion probes into bilateral IOs in ho17J mice. These mice with chronic implantation of microinfusion probes did not have worsening ataxia, measured by rotarod, suggesting that this procedure does not cause prominent injuries to IOs. We have done n = 3 experiments and we found that lidocaine microinfusion into bilateral IOs can real time suppress tremor and cerebellar oscillatory activities. After about 5 minutes of stopping lidocaine microinfusion, tremor and cerebellar oscillations returned gradually. These experiments indicate the essential role of activities of CFs, originated from IOs, in tremor.

Next steps: We will spend the next 3 months to increase the n number to achieve 10 mice for this experiment. We will also perform data analysis to assess tremor frequency and amplitude before, during, and after lidocaine microinfusion in the next 2 months for this aim.

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 worked with the Duke viral core to package AAV8-miniSOG. We also spent the past 4 months to fine-tune the protocol for stereotactic injection of AAV8-miniSOG to achieve adequate CF terminal expression. With our newly developed protocol, we could make ho17J mice have widely expressed miniSOG throughout the cerebellar cortex, and these mini-SOG puncta colocalized with VGlut2 puncta in the molecular layer, which indicates CF pre-synaptic terminal expression of miniSOG. To further test whether these miniSOG expression is functional, we performed cerebellar slice recording of PC activities. Upon shining the blue light to activate miniSOG, we observed 50% reduction of complex spikes, originated from CF-PC synaptic transmission. These data strongly support that our miniSOG-mediated approach can inhibit CF-PC synaptic activities.

Next steps: We will finally test how miniSOG-mediated CF-PC synaptic inhibition influences tremor. We will perform stereotactic surgery in 10 ho17J mice to achieve CF terminal expression as mentioned above. We will then create a chronic cranial window (a coverslip on top of the cerebellum) in these mice. We will perform light-mediated synaptic inhibition by shinning the blue light over the cerebellum in freely-moving ho17J mice. We will measure PC activities and tremor before and after the light-mediated inhibition. These experiments will provide reliable results whether CF-PC synaptic activities play an essential role in tremor.