A versatile cholera toxin conjugate for neuronal targeting and tracing

Abstract

Tracing of neurons plays an essential role in elucidating neural networks in the brain and spinal cord. Cholera toxin B subunit (CTB) is already widely used as a tracer although its use is limited by the need for immunohistochemical detection. A new construct incorporating non-canonical azido amino acids (azido-CTB) offers a novel way to expand the range and flexibility of this neuronal tracer. Azido-CTB can be detected rapidlyin vivofollowing intramuscular tongue injection by ‘click’ chemistry, eliminating the need for antibodies. Cadmium selenide/zinc sulfide (CdSe/ZnS) core/shell nanoparticles were attached to azido-CTB by strain-promoted alkyne-azide cycloaddition to make a nano-conjugate. Following tongue injections the complex was detectedin vivoin the brainstem by light microscopy and electron microscopyviasilver enhancement. This method does not require membrane permeabilization and so ultrastructure is maintained. Azido-CTB offers new possibilities to enhance the utility of CTB as a neuronal tracer and delivery vehicle by modification using ‘click’ chemistry.