Origin and timing of voltage-sensitive dye signals within layers of the turtle cerebellar cortex

Abstract

Optical recording techniques were applied to the turtle cerebellum to localize synchronous responses to microstimulation of its cortical layers and reveal the cerebellum's three-dimensional processing. The in vitro yet intact cerebellum was first immersed in voltage-sensitive dye and its responses while intact were compared to those measured in thick cerebellar slices. Each slice is stained throughout its depth, even though the pial half appeared darker during epi-illumination and lighter during trans-illumination. Optical responses were shown to be mediated by the voltagesensitive dye because the evoked signals had opposite polarity for 540-and 710-nm light, but no response to 850-nm light.Molecular layer stimulation of the intact cerebellum evoked slow transverse beams. Similar beams were observed in the molecular layer of thick transverse slices but not sagittal slices. With low currents, beams in transverse slices were restricted to sublayers within the molecular layer, conducting slowly away from the stimulus site. These excitatory beams were observed nearly all the way across the turtle cerebellum, distances of 4-6 mm. Microstimulation of the granule cell layer of both transverse or sagittal slices either evoked a local membrane depolarization restricted to a radial wedge, but these radial responses did not activate measurable molecular layer beams in transverse slices.White matter microstimulation in sagittal slices (near the ventricular surface of the turtle cerebellum) activated the granule cell and Purkinje cell layers, but not the molecular layer. These responses were nearly synchronous, were primarily caudal to the stimulation, and were blocked by cobalt ions. Therefore, synaptic responses in all cerebellar layers contribute to optical signals recorded in intact cerebellum in vitro (Brown and Ariel, 2009). Rapid radial signaling connects a sagittally-oriented, fast-conduction system of the deep layers with the transverse-oriented, slowconducting molecular layer, thereby permitting complex temporal processing between two tangential but orthogonal paths in the cerebellar cortex.

A novel path for rapid transverse communication of vestibular signals in turtle cerebellum

“…The details of this preparation and the optical recording methods have been presented in detail elsewhere (Brown and Ariel 2009;Fan et al 1993;Rosenberg and Ariel 1990;Ariel and Brown 2010). Briefly, the brain was removed from the anesthetized animal, its telencephalon was quickly ablated, and the brain stem was placed in a Petri dish of oxygenated physiological media.…”

“…A previous report (Ariel and Brown 2010) showed optical recordings of a slow beam of activation entirely within the ML from thick transverse slices. Although those findings were quite consistent, there was only limited success in this series of experiments in measuring a fast connection between the lateral edges of the Cb.…”

Current Practice in Using Voltage Imaging to Record Fast Neuronal Activity: Successful Examples from Invertebrate to Mammalian Studies

“…The third model used in the turtles’ brain for optical imaging is the intact cerebellum in vitro. This is convenient because the cerebellum in reptiles is thin and unfoliated, and its entire surface can be stained with VSD [ 163 , 164 , 165 ]. The vestibular stimulation in this model can be mimicked via the electric stimulation of VII cranial nerve or the inferior olive.…”