Introduction: There are currently no molecular techniques to visualize human cortical connectivity. Several in vivo methods (axonal tracing) have been developed but are limited to animal use only. Resected human cortical tissue from epileptic patients is potentially amenable to these techniques and can be used to study cortical connectivity. In this study we describe a rat model of ex vivo retrograde transport (RT) to characterize cortical connectivity. This model serves as the basis for axonal tracing in resected human cortical specimens.

Methods: Resected human temporal cortex and motor cortex (M1) or CA1 of 3mm coronal rat brain sections were injected with 100nL and 50nL 2% cholera-toxin subunit-b (CTB), respectively. Tissue was maintained at 25? and 32?C on a patch-clamp stage with continuously circulating carbogenated artificial cerebrospinal fluid (cACSF) (rate of 3mL/min or 6mL/min) for 4, 24, 48 and 96hrs. Tissue blocks were sectioned, stained and analyzed for axonal transport using cell counting, injection volume and length of axonal transport. In vivo rat studies were used to corroborate axonal transport. Tissue toxicity and ischemia were analyzed with H&E staining of the sections above.

Results: Resected tissue was successfully kept alive ex vivo, which allowed CTB transport to occur. This was confirmed with retro-2, a small molecule RT inhibitor. Longer incubation times, 32?C stage temperature and 6mL/min cACSF yielded a statistically significant increase in RT. At 72 and 96 hours, cells were traced to the contralateral M1 and thalamus with transport across the corpus callosum and thalamocortical radiations. CA1 injections yielded RT to CA3 along Schaffer’s collaterals in rat. Injected human tissue demonstrated specific cortico-cortico projections with RT and tagged cells in up to 1cm of tissue.

Conclusions: In this study we present a novel ex vivo method of axonal tracing in rat and human cortical tissue. Ex vivo RT is currently being used to define cortical structure in resected epileptic and normal human cortex.

Patient Care: Given current limitations of molecular techniques, basic human cortical connectivity has not been characterized. As such, the connectivity status in epileptic tissue is also unclear. This technique will help to characterize connectivity in epileptic tissue and will thus elucidate potential mechanisms for epileptogenesis and novel treatment paradigms.

Learning Objectives: By the conclusion of this session, participants should be able to: 1) Describe a new method for cortical connectivity tracing in human tissue 2) Discuss the clinical implications of this technique in epilepsy.

References: 1. Bohland, J.W., et al., A Proposal for a Coordinated Effort for the Determination of Brainwide Neuroanatomical Connectivity in Model Organisms at a Mesoscopic Scale. PLoS Comput Biol, 2009. 5(3): p. e1000334. 2. Pinskiy, V., et al., High-Throughput Method of Whole-Brain Sectioning, Using the Tape-Transfer Technique. PLoS One, 2015. 10(7): p. e0102363.