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Multiplex imaging relates quantal glutamate release to presynaptic Ca²⁺ homeostasis at multiple synapses in situ.

ABSTRACT: Information processing by brain circuits depends on Ca²⁺-dependent, stochastic release of the excitatory neurotransmitter glutamate. Whilst optical glutamate sensors have enabled detection of synaptic discharges, understanding presynaptic machinery requires simultaneous readout of glutamate release and nanomolar presynaptic Ca²⁺ in situ. Here, we find that the fluorescence lifetime of the red-shifted Ca²⁺ indicator Cal-590 is Ca²⁺-sensitive in the nanomolar range, and employ it in combination with green glutamate sensors to relate quantal neurotransmission to presynaptic Ca²⁺ kinetics. Multiplexed imaging of individual and multiple synapses in identified axonal circuits reveals that glutamate release efficacy, but not its short-term plasticity, varies with time-dependent fluctuations in presynaptic resting Ca²⁺ or spike-evoked Ca²⁺ entry. Within individual presynaptic boutons, we find no nanoscopic co-localisation of evoked presynaptic Ca²⁺ entry with the prevalent glutamate release site, suggesting loose coupling between the two. The approach enables a better understanding of release machinery at central synapses.

SUBMITTER: Jensen TP

PROVIDER: S-EPMC6441074 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Multiplex imaging relates quantal glutamate release to presynaptic Ca2+ homeostasis at multiple synapses in situ.

Jensen Thomas P TP Zheng Kaiyu K Cole Nicholas N Marvin Jonathan S JS Looger Loren L LL Rusakov Dmitri A DA

Nature communications 20190329 1

Information processing by brain circuits depends on Ca2+-dependent, stochastic release of the excitatory neurotransmitter glutamate. Whilst optical glutamate sensors have enabled detection of synaptic discharges, understanding presynaptic machinery requires simultaneous readout of glutamate release and nanomolar presynaptic Ca2+ in situ. Here, we find that the fluorescence lifetime of the red-shifted Ca2+ indicator Cal-590 is Ca2+-sensitive in the nano ...[more]

PMID: 30926781

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Project description:Nicotinic acid adenine dinucleotide phosphate (NAADP) is a second messenger that evokes calcium release from intracellular organelles by the engagement of calcium release channels, including members of the Transient Receptor Potential (TRP) family, such as TRPML1, the (structurally) related Two Pore Channel type 1 (TPC1) and TPC2 channels as well as Ryanodine Receptors type 1 (RYR1; Guse, 2012). NAADP evokes calcium release from acidic calcium stores of many cell types (Guse, 2012), and NAADP-sensitive Ca2+ stores have been described in hippocampal neurons of the rat (Bak et al., 1999; McGuinness et al., 2007). Glutamate triggers Ca2+-mediated neuronal excitotoxicity in inflammation-induced neurodegenerative pathologies such as Multiple Sclerosis (MS; Friese et al., 2014), and when applied extracellularly to neurons glutamate can elevate NAADP levels in these cells. Accordingly, glutamate-evoked Ca2+ signals from intracellular organelles were inhibited by preventing organelle acidification (Pandey et al., 2009). Analysis of reported RNA sequencing experiments of cultured hippocampal neurons revealed the abundance of Mcoln1 (encoding TRPML1), Tpcn1, and Tpcn2 (encoding TPC1 and TPC2, respectively) as potential NAADP target channels in these cells. Transcripts encoding Ryr1 were not found in contrast to Ryr2 and Ryr3. To study the contribution of NAADP signaling to glutamate-evoked calcium transients in murine hippocampal neurons we used the NAADP antagonists Ned-19 (Naylor et al., 2009) and BZ194 (Dammermann et al., 2009). Our results show that both NAADP antagonists significantly reduce glutamate-evoked calcium transients. In addition to extracellular glutamate application, we studied synchronized calcium oscillations in the cells of the neuronal cultures evoked by addition of the GABAA receptor antagonist bicuculline. Pretreatment with Ned-19 (50 μM) or BZ194 (100 μM) led to an increase in the frequency of bicuculline-induced calcium oscillations at the cost of calcium transient amplitudes. Interestingly, Ned-19 triggered a rise in intracellular calcium concentrations 25 min after bicuculline stimulation, leading to the question whether NAADP acts as a neuroprotective messenger in hippocampal neurons. Taken together, our results are in agreement with the concept that NAADP signaling significantly contributes to glutamate evoked Ca2+ rise in hippocampal neurons and to the amplitude and frequency of synchronized Ca2+ oscillations triggered by spontaneous glutamate release events.

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Multiplex imaging relates quantal glutamate release to presynaptic Ca²⁺ homeostasis at multiple synapses in situ.

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Multiplex imaging relates quantal glutamate release to presynaptic Ca<sup>2+</sup> homeostasis at multiple synapses in situ.

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