Validated multi‐step approach for in vivo recording and analysis of optogenetically evoked glutamate in the mouse globus pallidus

Abstract

Precise quantification of extracellular glutamate concentrations upon neuronal activation is crucial for the understanding of brain function and neurological disorders. While optogenetics is an outstanding method for the correlation between distinct neurons and their role in circuitry and behavior, the electrochemically inactive nature of glutamate has proven challenging for recording upon optogenetic stimulations. This difficulty is due to the necessity for using enzyme-coated microelectrodes and the risk for light-induced artifacts. In this study, we establish a method for the combination of in vivo optogenetic stimulation with selective measurement of glutamate concentrations using enzyme-coated multielectrode arrays and amperometry. The glutamatergic subthalamic nucleus (STN), which is the main electrode target site in deep brain stimulation treatment of advanced Parkinson's disease, has recently proven opotogenetically targetable in Pitx2-Cre-transgenic mice and was here used as model system. Upon stereotactic injection of viral Channelrhodopsin2-eYFP constructs into the STN, amperometric recordings were performed at a range of optogenetic stimulation frequencies in the globus pallidus, the main STN target area, in anesthetized mice. Accurate quantification was enabled through a multi-step analysis approach based on self-referencing microelectrodes and repetition of the experimental protocol at two holding potentials, which allowed for the identification, isolation and removal of photoelectric and photoelectrochemical artifacts. This study advances the field of in vivo glutamate detection with combined optogenetics and amperometric recordings by providing a validated analysis framework for application in a wide variety of glutamate-based approaches in neuroscience.

A disynaptic circuit in the globus pallidus controls locomotion inhibition

“…The STN is the only glutamatergic nucleus in the BG, and it represents the main excitatory input to GP neurons (Kita and Kitaï, 1987;Smith et al, 1990). It has been shown in mice that optogenetic activation of STN axon terminals within the GP increases glutamate release (Viereckel et al, 2018) and c-Fos expression (Tian et al, 2018). We show here that, similar to D2-SPN inputs, STN inputs differentially impact the activity of prototypic and arkypallidal neurons.…”

Optogenetic investigation into the role of the subthalamic nucleus in motor control

“…The Pitx2 gene encodes a transcription factor essential for STN development and function [26][27][28] . We recently verified that expression of the optogenetic ion channel Channelrhodopsin (ChR2) in the STN of Pitx2-Cre mice causes post-synaptic currents and glutamate release in STN target areas upon photostimulation 24,25 , thus validating the use of optogenetics in Pitx2-Cre mice for the study of STN.…”

“…By implementing optogenetics, new possibilities to directly compare the behavioral readout upon STN excitation and inhibition has opened up. Based on our previous data showing that 20 Hz photostimulation was sufficient to release glutamate in the GP of Pitx2/ChR2 mice 25 , a photoexcitation protocol designed for behavioral motor analysis was formulated. We found that GP neurons were excited already upon a 0. the open field test, immediate self-grooming was initiated upon STN excitation.…”

“…This finding verified functional connectivity between the STN and GP. Next, based on previous confirmation of glutamate release upon optogenetic excitation 25 , a photostimulation protocol designed to drive STN excitation in freely-moving animals was tested (Behavior protocol, Hz, 5 ms pulses, 5-8 mW; 100 seconds). Recordings in the GP identified increased frequency and firing rate of GP neurons for the whole duration of the stimulation, after which they returned to normal ( Figure 2G-H).…”

“…In this study, we aimed to tease out the impact of the STN on several movement parameters during non-pathological conditions. Based on our previous work validating optogenetic excitation of the STN using Pitx2-Cre mice to achieve selectivity 24,25 , protocols were here designed to allow direct comparison of the impact of optogenetic STN excitation and inhibition on motor output. Results now show that optogenetic excitation and inhibition gives rise to distinct mirror effects in several different motor parameters: STN inhibition enhances locomotion and reduces grooming; STN excitation reduces locomotion and induces grooming; STN excitation reduces balance and coordination.…”