Measurement of plasmalemmal dopamine transport, vesicular dopamine transport, and K+-stimulated dopamine release in frozen rat brain tissue

This report describes experiments designed to (1) establish the specificity of dopamine (DA) transporter (DAT)-mediated plasmalemmal DA transport, vesicular monoamine transporter-2 (VMAT-2)-mediated vesicular DA transport, and K+-stimulated DA release in samples prepared from frozen rat striata, and (2) characterize the time-course of the effects of freezing on these processes. The procedure described herein uses a simple method of freezing brain tissue (first cooling in ice-cold buffer and then freezing at -80 degrees C) that allows for the storage of rat striata followed by the assay of DA transport and K'-stimulated DA release using rotating disk electrode voltammetry. Plasmalemmal DA transport into samples prepared from frozen striata was blocked by the DAT inhibitor, cocaine, and vesicular DA transport was blocked by the VMAT-2 inhibitor, dihydrotetrabenazine. Additionally, K+-stimulated DA release was Ca+2-dependent. Freezing decreases DAT-mediated DA transport, VMAT-2-mediated DA transport, and K+-stimulated DA release. However activity is still measurable even after 3 weeks of storage. These results suggest that rat striata retain some DA transport and DA release activity even when stored frozen for a few weeks. Frozen storage of rat striata may thus be valuable for experiments requiring lengthy assays, the accumulation of material, or the transport of samples from one laboratory to another for analysis. These results may also be applicable to the study of frozen human brain tissue. (C) 2009 Elsevier B.V. All rights reserved.