Abstract
Calcium triggers dopamine release from presynaptic terminals of midbrain dopaminergic (mDA) neurons in the striatum. However, calcium transients within mDA axons and axon terminals are difficult to study and little is known about how they are regulated. Here we use a newly-developed method to measure presynaptic calcium transients (PreCaTs) in axons and terminals of mDA neurons with a genetically encoded calcium indicator (GECI) GCaMP3 expressed in transgenic mice. Using a photomultiplier tube-based system, we measured electrical stimulation-induced PreCaTs of mDA neurons in dorsolateral striatum slices from these mice. Single-pulse stimulation produced a transient increase in fluorescence that was completely blocked by a combination of N- and P/Q-type calcium channel blockers. DA and cholinergic, but not serotoninergic, signaling pathways modulated the PreCaTs in mDA fibers. These findings reveal heretofore unexplored dynamic modulation of presynaptic calcium in nigrostriatal terminals.
Highlights
The striatum has the richest DA innervation in the CNS, provided by the widely arborized axons of midbrain dopaminergic (mDA) neurons [1]
GCaMP3 expression in mDA neurons of PITX3/GC transgenic mice was visualized by co-immunostaining with specific antibodies against green fluorescent protein (GFP) and tyrosine hydroxylase (TH)
The GFP signals appeared in the cytosol of soma and processes, in both substantia nigra pars compacta (SNpc) and ventral tegmental area (VTA) mDA neurons, as well as in dorsal striatal DA axons and terminals (Fig. 1B)
Summary
The striatum has the richest DA innervation in the CNS, provided by the widely arborized axons of mDA neurons [1]. The ability to measure Ca2+ in DA terminals has been hampered by the small dimensions of DA axons and terminals, the massive distribution of DA axonal ramifications in the striatum [1], and difficulties associated with loading Ca2+ indicators selectively into DA axons/terminals while avoiding nearby cellular elements. Encoded Calcium Indicator (GECIs) based on chimeric fluorescent proteins allow investigators to avoid many limitations of conventional small-molecule Ca2+ dyes [6]. They can be targeted for expression in specific cell types, avoiding indiscriminate loading. The utility of GCaMPs for measuring presynaptic Ca2+ has not been widely assessed [8,9]
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