Abstract
Damage to one vestibular apparatus (unilateral vestibular deafferentation, UVD) results in severe postural and ocular motor disturbances (such as spontaneous nystagmus, SN) that recover over time in a process known as vestibular compensation. However, the underlying neurochemical mechanisms of vestibular compensation are poorly understood. While UVD affects many areas in the CNS, attention has focused upon the partially deafferented second order neurons in the vestibular nuclei complex (VNC). Several converging lines of evidence suggest that Ca²⁺-permeable ion channels (N-methyl-D-aspartate receptors and L-type voltage-gated Ca²⁺-channels) and intracellular Ca²⁺-dependent protein kinases play an important role in vestibular compensation. However, the nature of this involvement and the locus of these changes are unknown. The aim of this thesis was to investigate the role of Ca²⁺ signalling pathways in the VNC during vestibular compensation in guinea pig. These issues were investigated in three separate experiments that utilised two methodological approaches: i) in vitro assays were used to determine the nature and extent of protein phosphorylation within the VNC at various stages of compensation; and ii) ion channel blockers or cell-permeable kinase inhibitors were injected directly into the VNC immediately before UVD to determine whether or not these systems were causally involved in compensation.
The results of experiment 1 (Chapter 5) showed that a bolus intra-VNC injection of an uncompetitive NMDA receptor antagonist, but not an L-type voltage-gated Ca²⁺ channel antagonist, temporarily reduced SN frequency at the earliest measurement time (6 hours post-UVD). These results suggested that the initial expression of SN required, in part, the activation of NMDA receptors in the VNC on the side of the UVD, and by inference, Ca²⁺ entry through the ion channel. The results of experiment 2 (Chapter 6) revealed that the medial VNC contains abundant Ca²⁺/calmodulin-dependent and Ca²⁺/phospholipid-dependent protein kinase activities. The same VNC tissue removed from animals at various times after UVD, showed that vestibular compensation is accompanied by specific changes in the phosphorylation of several major protein kinase C substrates. These included an unidentified 46-kDa band, and a 75-kDa band with similar characteristics to the myristoylated alanine-rich C kinase substrate (MARCKS). These results suggest that protein kinase C signalling pathways may be involved in vestibular compensation. The results of experiment 3 (Chapter 7) are consistent with these results showing that intra-VNC infusion of a protein kinase C inhibitor, but not a Ca²⁺/calmodulin-dependent protein kinase II inhibitor, significantly increased SN at the earliest measurement times (6 and 8 hours), but had no effect upon the time taken to achieve compensation or on postural compensation. These results suggest that the induction of SN compensation involves protein kinase C activity in the VNC. Taken together, these findings suggest that the mechanisms underlying the expression of SN (e.g., Ca²⁺ influx via NMDA receptors) are possibly distinct from those that initiate its compensation (e.g., PKC activation). The downstream effects of raised intracellular Ca²⁺ may involve protein kinase C-dependent phosphorylation of key intracellular proteins that initiate long-lasting changes in cellular function within the VNC.