Post-surgical MRI scans were used to reassess the chamber orientation and select the chamber positions suitable for the electrode approach to the FOR. Neuronal activity recorded from physiological landmarks such as the brainstem oculomotor nuclei helped to double check the orientation of the recording chamber relative to the FOR, allowing us to optimize the approach of the glass-coated tungsten microelectrodes (impedances 1-2 MΩ; Alpha Omega Engineering, Nazareth, Israel) to the FOR. A successful electrode approach to the FOR was characterized by a characteristic sequence of background activity along a tract: first, the appearance of the hallmarks of cerebellar grey matter activity like the dense granule cell background activity and the occurrence of complex spikes; then a longer stretch of a silent background reflecting the passage through white matter separating cerebellar cortex and the fastigial nucleus; and finally, when entering the fastigial nucleus, again relatively dense background activity along with the occurrence of characteristic saccaderelated burst firing in its caudal part and the absence of complex spikes.
Glass coated tungsten microelectrodes
Manufactured by Omega Engineering
Sourced in Israel
Glass-coated tungsten microelectrodes are precision laboratory instruments used for recording electrical signals in small, localized areas. They are composed of a tungsten wire core coated with a thin layer of glass, providing a highly durable and insulated recording tip. These electrodes are designed for use in a variety of experimental applications requiring the measurement of electrical activity at the cellular or tissue level.
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4 protocols using glass coated tungsten microelectrodes
1
Targeting the Fastigial Oculomotor Region
2
Extracellular Recordings from Purkinje Cells in Oculomotor Vermis
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We performed extracellular recordings from PCs using glass-coated tungsten microelectrodes (impedance: 1 to 2 MΩ) that were purchased from Alpha Omega Engineering, Nazareth, Israel. The position of the electrodes, which were targeted toward the OMV, was controlled using a modular multielectrode manipulator (Electrode Positioning System and Multi-Channel Processor, Alpha Omega Engineering). The identity and the exact coordinates of the OMV predicted by the MRI scans were confirmed by physiological criteria, i.e., the presence of a dense saccade-related background activity, reflecting multiunit granule cells activity. To differentiate action potentials from the underlying LFP signals, extracellular potentials, recorded at the sampling rate of 25 KHz, were high band-pass filtered (300 Hz to 3 KHz) and low-pass filtered (30 Hz to 400 Hz), respectively. A total of 160 PCs were recorded out of which 151 were considered for analysis. A table summarizing the total number of PCs recorded from each monkey, either in left, right, or both directions, can be found in S1 Table .
3
Extracellular Recording of Purkinje Cell Action Potentials
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Action potentials of PCs were recorded extracellularly using glass-coated tungsten microelectrodes (1–2 MΩ impedance at 1 kHz; Alpha Omega Engineering, Nazareth, Israel) advanced with an 8-probe electrode system (Alpha Omega Engineering, Nazareth, Israel). In most cases we used maximally 4 electrodes, arranged linearly either along the rostrocaudal or the medio-lateral axis and separated by 2 mm each. We approached the OMV by using the stereotaxic coordinates provided by the anatomical MRI scans and identified the OMV by resorting to well established criteria, namely the dense saccade-related granule cell background and the appearance of saccade-related single units in the neighbouring layers. The electrode signal was band-pass filtered for frequencies from 300 to 3000 Hz to enable the isolation of spikes. SS and CS were detected online by using a Multi Spike Detector (Alpha Omega Engineering, Nazareth, Israel) which detects and sorts spikes according to the features of template waveforms.
4
Extracellular Recordings in Oculomotor Vermis
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Extracellular recordings with commercially available glass-coated tungsten microelectrodes (impedance: 1-2 MΩ; Alpha Omega Engineering, Nazareth, Israel) were performed using a modular multi-electrode manipulator (Electrode Positioning System and Multi-Channel Processor, Alpha Omega Engineering) whose position was estimated, based on the position and orientation of the chamber relative to the brain, using a stereotactic apparatus and later confirmed by post-surgical MRI scans. Saccade-related modulation of an intense background activity, reflecting multi-unit granule cell activity, paralleled by saccade-related modulation in the local field potential record (LFP, <150 Hz bandwidth) served as electrophysiological criteria for identifying the OMV (Fig. 1A, middle). Extracellular potentials, sampled at 25 KHz, were high band-pass (300 Hz -3 KHz) and low-pass filtered (<150 Hz) to differentiate PC action potentials and LFP signals, respectively (Fig. 1A, bottom).
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