Zinc-enriched neurons in the spinal cord, cerebellum and peripheral sympathetic system

Abstract: Zinc ions are abundant in the nervous system and are involved in many biological functions. Most zinc is tightly bound to macromolecules, whereas a small fraction of zinc exists in synaptic vesicles of the zinc-enriched (ZEN) neurons, and can be visualized using various histochemical techniques, including autometallography (AMG). Recent studies have demonstrated that a zinc-specific transporter, zinc transporter 3 (ZnT3), is located on the membrane of a pool of synaptic vesicles, and transports zinc into synaptic vesicles. The densest populations of ZEN terminals are found in the forebrain, including the olfactory bulb, hippocampus, amygdala and neocortex, where zinc is present in presynaptic vesicles of a subset of glutamatergic neurons. In vitro experiments have shown that free zinc ions can modulate the functions of several postsynaptic receptors, including N-methyl-D-aspartate (NMDA), a-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), g-aminobutyric acid (GABA) and glycine receptors. The aims of the present study were to evaluate the presence of inhibitory ZEN neurons in the spinal cord and the cerebellum, and to clarify whether there are zinc-containing neurons in the peripheral nervous system.In the spinal cord, confocal laser scanning microscopy proved the existence of ZnT3-containing GABAergic terminals in all laminae of the spinal gray matter, and the lateral and ventral funiculi of the white matter. Ultrastructurally, colocalization of zinc ions and glutamic acid decarboxylase (GAD) immuno-reactivity were seen in a pool of presynaptic terminals in the above locations. Furthermore, the regional distribution of ZEN neuronal somata in the spinal cord was confirmed using the retrograde tracing AMG technique. In the cerebellum, terminals colocalized with ZnT3 and GAD were predominantly distributed in the granule cell layer. Based on the size and position, these double labelled elements are suggested to be axonal terminals of the Golgi and basket/stellate cells, in the granular and molecular layers, respectively. The present results therefore indicate that a zinc-containing GABAergic system exists in the spinal cord and cerebellum, and that synaptic zinc ions are probably co-released with GABA in a subpopulation of GABAergic terminals, modulating GABA receptors of the postsynaptic neurons. In a stop-flow sciatic nerve crush animal model, a time-dependent accumulation of zinc ions and ZnT3 could be identified both proximally and distally to the crush sites. Electron microscopic data proved that the zinc ions were predominantly present in unmyelinated axons. Double labelling of ZnT3 and other neuronal markers showed that ZnT3 colocalized well with tyrosine hydroxylase (TH), but not with vesicular acetylcholine transporter (VAChT), calcitonin gene-related peptide (CGRP) or neuropeptide Y (NPY). A small population of post-ganglionic sympathetic neurons in lumbar sympathetic ganglia were ZnT3-positive or contained zinc ions. Taken together, these results suggest that there is a zinc-containing sympathetic system in the peripheral nervous system. The rapid appearance of zinc ions in crushed sciatic axons both proximally and distally to the crush indicates that endogenous zinc ions undergo fast antero- and retrograde transport in ZEN neuronal pathways.