Methods for studying synaptosomal copper release

J Neurosci Methods. 2003 Sep 30;128(1-2):159-72. doi: 10.1016/s0165-0270(03)00173-0.


Cu is thought to play an important role in the pathogenesis of several neurodegenerative diseases, such as Wilson's, Alzheimer's, and probably in prion protein diseases like Creutzfeld-Jakob's disease. Until now, no method existed to determine the concentration of this cation in vivo. Here, we present two possible approaches combined with a critical comparison of the results. The successful use of fluorescent ligands for the determination of Ca2+-concentrations in recent years encouraged us to seek a fluorophore which specifically reacts to Cu2+ and to characterize it for our purposes. We found that the emission of TSPP (tetrakis-(4-sulfophenyl)porphine) at an emission wavelength of 645 nm is in vitro highly specific to Cu2+ (apparent dissociation constant Kd=0.43 +/- 0.07 microM at pH 7.4). It does not react with the most common divalent cations in the brain, Ca2+ and Mg2+, unlike most of the other dyes examined. In addition, Zn2+ quenches TSPP fluorescence at a different emission wavelength (605 nm) with a Kd of 50 +/- 2.5 microM (pH 7.0). With these findings, we applied the measurement of Cu with TSPP to a biological system, showing for the first time in vivo that there is release of copper by synaptosomes upon depolarisation. Our findings were validated with a completely independent analytical approach based on ICP-MS (inductively-coupled-plasma mass-spectrometry).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cattle
  • Cell Membrane Permeability / physiology
  • Chromaffin Cells / chemistry
  • Chromaffin Cells / metabolism
  • Copper / analysis*
  • Copper / physiology*
  • Mass Spectrometry / methods
  • Porphyrins / analysis
  • Synaptosomes / chemistry*
  • Synaptosomes / metabolism*


  • Porphyrins
  • tetraphenylporphine sulfonate
  • Copper