Genetically restricted metabolic labeling in Danio rerio, a simple vertebrate capable of protein synthesis-dependent memory formation.

摘要

Determining which neural circuits and proteins are involved in encoding memories is a central goal in neuroscience. Protein expression in the nervous system is known to undergo regulated changes in response to changes in behavioral states, in particular long-term memory formation. In this study we developed tools to investigate protein synthesis in an intact organism, the larval zebrafish, capable of simple learning behavior. Methods have recently been developed (BONCAT and FUNCAT), which introduce noncanonical amino acids bearing small bioorthogonal functional groups into proteins using the cells' own translational machinery. Using the selective 'click reaction', this allows for the identification and visualization of newly synthesized proteins in vitro..;Here we demonstrate that noncanonical amino acid labeling can be achieved in vivo in the larval zebrafish. We show that azidohomoalanine is metabolically incorporated into newly synthesized proteins, in a time- and concentration-dependent manner, but has no apparent toxic effect and does not influence simple behaviors such as spontaneous swimming and escape responses. This enables fluorescent labeling of newly synthesized proteins in whole mount larval zebrafish. Furthermore, we demonstrate that genetically restricted expression of a mutant methionyl-tRNA synthetase permits cell-specific metabolic labeling with the larger noncanonical amino acid, azidonorleucine, both in vitro and in vivo. Finally, we present an associative conditioning paradigm for larval zebrafish. During a three-hour training period, 6-8dpf larvae learn to associate the social reward of visual access to a group of conspecifics with a dark environment. The memory formed during this place-conditioning paradigm undergoes rapid extinction, but is extremely stable, lasting for up to 36h. Furthermore, memory formation is both protein synthesis- and partially NMDAR-dependent. Together, the techniques developed in this study will enable the investigation of protein synthesis during long-term memory formation in the larval zebrafish.