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

Abstract

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.