A technique for ¹²⁵I-labelling of neurotrophins and the use of retrograde axonal transport as a bioassay

The protocol presented here details a technique which enables the neurotrophins nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin- 4 (NT-4), and brain-derived neurotrophic factor (BDNF) to be labelled using ¹²⁵I-labelling of neurotrophic factors is the IODO-GEN method. Following the iodination of neurotrophins it must be established that the labelling procedure has not affected the biological activity of the protein. Traditional methods of assaying the bioactivity of ¹²⁵I-labelled neurotrophins have several disadvantages and a much easier protocol to use is the retrograde axonal transport of these proteins in sympathetic and sensory neurons of adult mice. High specific activity ¹²⁵I-labelled neurotrophin, to which known amounts of unlabelled neurotrophin are added, is injected into the right anterior eye chamber of adult mice under anaesthetic and the animals are left to recover for 16 h, after which they are sacrificed and both superior cervical ganglia (SCG) and trigeminal ganglia (TGG) are removed. The accumulated radioactivity in each ganglion is determined using a γ-counter on the injected side. By comparing the amount of protein injected with the amount transport, the specific activity of the bioactive labelled neurotrophin can be determined.