High-resolution analysis of bound Ca²⁺ in neurons and synapses

Calcium (Ca²⁺) is a well-known second messenger in all cells, and is especially relevant for neuronal activity. Neuronal Ca²⁺ is found in different forms, with a minority being freely soluble in the cell and more than 99% being bound to proteins. Free Ca²⁺ has received much attention over the last few decades, but protein-bound Ca²⁺ has been difficult to analyze. Here, we introduce correlative fluorescence and nanoscale secondary ion mass spectrometry imaging as a tool to describe bound Ca²⁺ As expected, bound Ca²⁺ is ubiquitous. It does not correlate to free Ca²⁺ dynamics at the whole-neuron level, but does correlate significantly to the intensity of markers for GABAergic pre-synapse and glutamatergic post-synapses. In contrast, a negative correlation to pre-synaptic activity was observed, with lower levels of bound Ca²⁺ observed in the more active synapses. We conclude that bound Ca²⁺ may regulate neuronal activity and should receive more attention in the future.