Motor learning in the VOR: the cerebellar component

Abstract

This paper reviews results that support a model in which memory for VOR gain is initially encoded in the flocculus, and in which cerebellar LTD and LTP are responsible for gain increases and gain decreases, respectively. We also review data suggesting that after it is encoded, motor memory can either be disrupted, possibly by a local mechanism, or else consolidated. We show that consolidation can be rapid, in which case the frequency dependence of learning is unchanged and we will argue that this is consistent with a local mechanism of consolidation. In the longer term, however, the available evidence supports the transfer of memory out of the flocculus. In new experiments reported here, we address the mechanism of memory encoding. Pharmacological evidence shows that both mGluR1 and GABA_B receptors in the flocculus are necessary for gain-up, but not for gain-down learning. Immunohistochemical experiments show that the two receptors are largely segregated on different dendritic spines on Purkinje cells. Together with what is already known of the mechanisms of cerebellar LTD and LTP, our data suggest that the direction of learning may be determined by interactions among groups of spines. Our results also provide new evidence for the existence of frequency channels for vestibular signals within the cerebellar cortex.

Appendix

Methods for immunohistochemistry

Two adult C57/Bl6 mice were deeply anesthetized with ketamine (75 mg/kg) and xylazine (5 mg/kg) before transcardial perfusion with 0.1 M PBS followed by fixative (4% para-formaldehyde containing 15% picric acid, pH 7.40). The brains were removed, bisected sagittally along the midline, and postfixed for an additional 2 h at room temperature. The brains were then transferred to a citrate buffer (pH 4.5) and stored overnight at 4°C. Next, brains were irradiated in a microwave oven, on low power, for 3 min in fresh citrate buffer before cryoprotection in 10% DMSO for 3 h at room temperature. Finally, the brains were flash frozen and embedded in O.C.T. compound (Sakura-Finetek, USA) before sagittal sectioning of 30-μm sections.

Unmasking of GABA_B epitopes was accomplished by the treatment of the free-floating sections with porcine pepsin (Sigma; 0.5 mg/ml in 0.2 N HCl) for 3 min at 37°C (Nagy et al. 2004). The sections were incubated in a blocking solution containing 5% goat serum (Sigma) and 0.3% Triton X-100 for 1 h at room temperature before overnight incubation at 4°C in primary antibodies diluted in blocking solution. After washing in 0.1 M PBS, the sections were incubated for 2 h with secondary antibodies diluted in 5% goat serum at room temperature and then washed again in 0.1 M PBS before being mounted in ProLong Gold Antifade (Molecular Probes, Invitrogen). Visualization of immunostaining was done on a Zeiss LSM 510 confocal microscope. Co-localization was analyzed using object-based methods in the JACoP plugin (Bolte and Cordelieres 2006) for Image J, version 1.43u (NIH).

Anti-GABA_B R1 and R2 (1:100) antibodies were purchased from Neuromab. The anti-mGluR1a-specific antibody was generated as previously described (Hampson et al. 1994), and the secondary goat anti-mouse DyLight 488 (1:1,000) and goat anti-rabbit DyLight 549 (1:1,000) antibodies were purchased from Jackson Immunoresearch.

Three 100× images were taken from each of the two mice and within each of those three images, another four were taken at 500× and averaged. Each 100× field of view was counted as an independent sample.