Quantitative analysis of granule cell axons and climbing fiber afferents in the turtle cerebellar cortex

Abstract

The turtle cerebellar cortex is a single flat sheet of gray matter that greatly facilitates quantitative analysis of biotylinated dextran amine labeled granule cell and olivocerebellar axons and Nissl-stained granule and Purkinje neurons. On average, ascending granule cell axons are relatively thicker than their parallel fiber branches (mean +/- SD: 0.84 +/- 0.17 vs 0.64 +/- 0.12 microm, respectively). Numerous en passant swellings, the site of presynaptic contact, were present on both ascending and parallel fiber granule cell axons. The swellings on ascending axons (1.82 +/- 0.34 microm, n = 52) were slightly larger than on parallel fibers (1.43 +/- 0.24 microm, n = 430). In addition, per unit length (100 microm) there were more swellings on ascending axons (11.2 +/- 4.2) than on parallel fibers (9.7 +/- 4.2). Each parallel fiber branch from an ascending axon is approximately 1.5 mm long. Olivocerebellar climbing fiber axons followed the highly tortuous dendrites of Purkinje cells in the inner most 15-20% of the molecular layer. Climbing fibers displayed relatively fewer en passant swellings. The spatial perimeter of climbing fiber arbors (area) increased 72% from anteriorly (1797 microm2) to posteriorly (3090 microm2) and 104% from medially (1690 microm2) to laterally (3450 microm2). Differences in the size and spacing of en passant swellings on granule cell axons suggest that ascending axons may have a functionally more significant impact on the excitability of a limited number of radially overlying Purkinje cells than the single contacts by parallel fiber with multiple orthogonally aligned Purkinje cell dendrites. The spatially restricted distribution of climbing fibers to the inner most molecular layer, the paucity of en passant swellings, and different terminal arbor areas are enigmatic. Nevertheless, these finding provide important anatomical information for future optical imaging and electrophysiological experiments.

Topography and Response Timing of Intact Cerebellum Stained With Absorbance Voltage-Sensitive Dye

“…Conduction velocities of mammalian parallel fibers are 0.18 -0.27 m/s, although their lengths are not proportional to brain size. In turtles, parallel fibers have a mean diameter of 0.64 Ϯ 0.12 m and have been observed to transverse nearly the entire Cb (Tolbert et al 2004), forming roughly 100 synapses every millimeter. The optical responses following ML stimulation are consistent with continuous activation of the Cb along a narrow transverse beam from lateral edge to lateral edge.…”

“…The repeating modules of Cb each receive excitatory input from mossy fibers, which in turn stimulate granule cells (Walsh et al 1972). Axons from granule cells ascend to the ML synapse with PCs during the ascent and bifurcate to form parallel fibers (Huang et al 2006;Tolbert et al 2004). Stimulating mossy fibers activates a local patch of PCs via ascending axons of granule cells (Bower and Devor 2003;Cohen and Yarom 1998;Rokni et al 2007), as shown by correlated activity of granule cells and their overlying PCs (Lu et al 2005).…”

“…It has three layers overlying white matter whose sole output neurons, Purkinje cells (PCs), are excited by the ascending axons of the granule cells (Cohen and Yarom 1998;Huang et al 2006;Isope and Barbour 2002;Tolbert et al 2004), parallel fibers (Eccles et al 1966b), and climbing fibers (Eccles et al 1966a), which carry signals from the contralateral inferior olive (IO) (Dow 1942). Recent hypotheses have focused on Cb's role in behavioral timing (Kistler et al 2000;Kitazawa and Wolpert 2005;Xu et al 2006;Yarom and Cohen 2002), although controversy surrounds reports that olivocerebellar responses (Sugihara et al 1993) are synchronous (see Aggelopulos et al 1995;Baker and Edgley 2006a,b and the responses of Lang et al 2006).…”

Topography of Purkinje Cells and Other Calbindin-Immunoreactive Cells Within Adult and Hatchling Turtle Cerebellum

“…However, unlike in most other species, the turtle cerebellar cortex is relatively small and unfoliated, a morphological feature that permits physiological [6][7][8][9] and anatomical studies [5,21,29,30] to answer fundamental questions of cortical structure and function. Here, the total Purkinje cell number in the cerebellum counts were not estimated, but rather were counted from the entire cerebellum from serial reconstructions using calbindin immunohistochemistry.…”

“…Its cerebellum (Cb) is uniquely suited for a topographic analysis because it is an unfoliated sheet of cortex covering the fourth ventricle, unlike most vertebrates whose cerebella is highly foliated. In spite of this simplification, the turtle Cb retains the same cellular organization of cerebella of higher vertebrates [5], consisting of a molecular layer, a Purkinje cell (PC) layer, and an internal granule cell layer. It is connected to each side of the brainstem by a broad attachment to the optic tectum and the dorsolateral part of the rostral medulla.…”