Presynaptic evidence for zinc release at the mossy fiber synapse of rat hippocampus

Vesicular zinc (Zn2+) is found in a subset of glutamatergic nerve terminals throughout the mammalian forebrain and is colocalized with glutamate. Despite well-documented neuromodulatory roles, exocytosis of endogenous Zn2+ from presynaptic terminals has never been directly demonstrated, because existing studies have measured elevated Zn2+ concentrations by examining the perfusate. Thus, the specific origin of synaptic Zn2+ remains a controversial subject. Here, we describe synaptic Zn2+ trafficking between cellular compartments at hippocampal mossy fiber synapses by using the fluorescent indicator Zinpyr-1 to label the hippocampal mossy fiber boutons. We determined endogenous Zn2+ exocytosis by direct observation of vesicular Zn2+ as decreasing fluorescence intensity from presynaptic axonal boutons in the stratum lucidum of CA3 during neural activities induced by the stimulation of membrane depolarization. This presynaptic fluorescence gradually returned to a level near baseline after the withdrawal of moderate stimulation, indicating an endogenous mechanism to replenish vesicular Zn2+. The exocytosis of the synaptic Zn2+ was also dependent on extracellular Ca2+ and was sensitive to Zn2+-specific chelators. Vesicular Zn2+ loading was sensitive to the vacuolar-type H+-ATPase inhibitor concanamycin A, and our experiments indicated that blockade of vesicular reloading with concanamycin A led to a depletion of that synaptic Zn2+. Furthermore, synaptic Zn2+ translocated to the postsynaptic cell body upon release to produce increases in the concentration of weakly bound Zn2+ within the postsynaptic cytosol, demonstrating a feature unique to ionic substances released during neurotransmission. Our data provide important evidence for Zn2+ as a substance that undergoes release in a manner similar to common neurotransmitters. (C) 2007 Wiley-Liss, Inc.