Lanthanide probes for labelling the dopaminergic cells.

Sharon Muzerengi1, Manuel Tropiano2, Stephen Faulkner2, Keith Brain1. 1University of Birmingham, Birmingham, UK, 2University of Oxford, Oxford, UK

Background: Lanthanide conjugates are now widely used as cellular probes and imaging contrast agents by exploiting their fluorescence and magnetic properties1. Current imaging techniques for dopaminergic cell loss in degenerative parkinsonism use radioactive tracers2, which may not be widely available. Utility of lanthanide probes in imaging the dopaminergic system has not been explored before.

Aim: To determine if a lanthanide based probe can be used to label the dopamine transporter (DAT) in rat adrenal phaechromocytoma (PC12) cells.

Methods: Cellular uptake of Europium-Dopamine (Eu-DA) was measured in undifferentiated and differentiated PC12 cells using fluorescence microscopy. PC12 cells were obtained as a gift from A. Logan’s Laboratory at the University of Birmingham. Cells were plated onto poly-L-lysine coated coverslips (cell density= 50 000 cells per coverslip), placed in an organ bath and perfused with physiological saline followed by Eu-DA (300ᶙM) perfusion for 60minutes. PC12 cells and Eu-DA were excited with a mercury arc lamp light source at 390nm and a 605 emission filter was used. Images were taken at time 0min then every 10minutes up to 60minutes using a camera attached to an epifluorescence microscope. Eu-DA cellular uptake studies were repeated in the presence of GBR 12909 hydrochloride (1µM) a DAT blocker and desipramine 1ᶙM, a noradrenaline transporter (NET) blocker. To provide an internal control, the study protocol was revised so that uptake was assessed twice within the same experiment. Image analysis was performed using image J version. Regions of interest (RIO) were drawn around each cell. The RIO were automatically fitted onto subsequent images for each cell on the image stacks and mean cell fluorescence was measured. The rate of uptake for each experiment was calculated. Data was represented as mean ± SEM. Comparisons between groups were performed using paired and unpaired Student t-test or Mann-Whitney U test accordingly.

Results: Eu-DA cellular uptake in undifferentiated PC12 cells increased in a time dependent manner over 1 hour. However, rate of uptake was weak mean 0.08± 0.01, n=40cells. Wilcoxon signed rank test showed no difference in the rate of uptake between the 2 uptake stages when the revised protocol was used, median difference 0.0208 (P<0.05, n=40). Mann-Whitney U test showed no difference in the rate of Eu-Da uptake in the absence and presence of GBR and desipramine, median difference 0.0250 (p<0.05, n=50)

Conclusion: This study was unable to demonstrate DAT dependent Eu-DA uptake by PC12 cells. Further cellular uptake studies are required using lanthanide probes of greater luminescence to determine if the cellular uptake is DAT mediated.

References

1. Heffern, M. C. et al. (2014) Chem Rev 114: 4496-539

2. Cummings J.L et al. (2011).Brain 134: 3146-66