A method for the evaluation of intracranial tetrodotoxin injections

Tetrodotoxin is one of the most potent and oldest known neurotoxins. It acts by blocking the voltage-gated sodium channels in nerve cell membranes, leading to a transient silencing of neural activity. TTX is among the most widely used pharmacological agents for the temporary and selective blocking of neural structures. As such, an exact knowledge of the spatial and temporal diffusion gradient of TTX is important when planning pharmacological interventions. Here we report a method for the direct assessment of spatio-temporal TTX diffusion gradients using immunohistochemistry. TTX injections were performed in vivo via chronically implanted guiding cannulae, placed bilaterally in the dorsal entopallium of pigeons. To determine the temporal spread, animals were perfused at different time points after TTX injections. For visualization of the TTX affected area an immunohistochemical protocol was developed. The extension of staining was assessed 1 h after injection when TTX was diffused over 3 mm in all directions. TTX immunolabeling could be detected for up to 32 h; after 48 h no staining was found. Our findings provide a better understanding of the temporal decay and spread of intracranial TTX injections, thereby allowing a more reliable estimation of size and duration of TTX-effects.