Training and fucose metabolism in chick brain

Abstract

This thesis reports an extensive study on the changes in fucose metabolism when chicks are trained on a passive avoidance learning paradigm. The study is based on the analysis of an observed increase in fucose incorporation observed in vivo (Sukumar et al 1980). Experiments were conducted to determine the mechanism of this increase and an in vitro fucosylation system was established to isolate the specific glycoproteins involved. Finally a series of experiments were performed to investigate the exact nature of this training-related increase in fucosylation by interventive methodology. Chapter 1 introduces the extensive subject of learning and memory mainly from a physiological view, touching briefly on some of the major psychological classifications. Chapter 2 is a review of the physiological literature on learning and memory in the chick, covering two behavioural paradigms: imprinting and a passive avoidance learning task. It covers the basic anatomy and function of the avian telencephalon, and finally introduces the topic of glycoprotein structure and function. Chapter 3 is the first experimental Chapter, and covers the analysis of one of the enzymes in the metabolic pathway of fucose incorporation into glycoproteins: fucokinase. This Chapter documents the assay procedure, the physiological conditions for maximum enzyme activity and finally the effects of training on fucokinase activity. Activity is not regulated by increases in the physiological concentration of calcium or c'AMP or c'GMP. Maximum activity is elicited with a specific concentration of magnesium, 3.3mM. One hour and six hours after training there is a significant increase in fucokinase activity, which is located to the left base and right forebrain roof respectively. An extensive discussion of the experimental results and hypotheses are presented. Chapter 4 covers the experimental procedure of obtaining an active fucosylation system in chick forebrain slices in vitro. When chick forebrain slices are cut on a Mcllwain tissue chopper to a thickness of 0.4 mm, and incubated in a Hepes buffer pH 7.4 for 3 hr at 42 C, in the presence of labelled fucose an active fucosylation system is maintained. Fucose incorporation rates as high as 37 nmole/g pfot/hr were observed. Slices of forebrain tissue from (M) trained and (W) control chicks were analysed for fucose incorporation in vitro at specific times after training. The increase in fucose incorporation after training which was observed in vivo (Sukumar et al 1980) was replicated in vitro. The increase was localized to the right forebrain base. On subcellular fractionation the increase in fucose incorporation was found in the P3 fraction. An extensive study using gel electrophoresis of the P3 fraction indicated that there was greater labelling of proteins with molecular weights 12GK - 82K in the trained chicks compared to controls. The experimental findings are discussed in relation to previous in-vivo work. Chapter 5 is concerned with the question of the exact nature of the training-related increase in fucose incorporation. Experiments were devised to determine if the increase was an independent increase in fucose incorporation or, alternatively, if it was associated with an increase in protein synthesis de novo. A series of four experiments are outlined analysing both the effects of cycloheximide and saline on fucose and leucine incorporation in vivo and in vitro in trained and control chicks. The training-related increase in fucose incorporation was abolished when cycloheximide was injected intracranially, which would suggest that the increase in fucosylation was occurring on protein synthesis de novo. The results of these experiments are discussed in relation to the time after training during which protein synthesis is important in memory formation. Chapter 6 is a general discussion of all the experiments and attempts to substantiate and rationalize a link between all the observed results. Chapter 7 covers a number of experiments for future work.