Real-Time Imaging Reveals Augmentation of Glutamate-Induced Ca2+ Transients by the NO-cGMP Pathway in Cerebellar Granule Neurons

Abstract

Dysfunctions of NO-cGMP signaling have been implicated in various neurological disorders. We have studied the potential crosstalk of cGMP and Ca2+ signaling in cerebellar granule neurons (CGNs) by simultaneous real-time imaging of these second messengers in living cells. The NO donor DEA/NO evoked cGMP signals in the granule cell layer of acute cerebellar slices from transgenic mice expressing a cGMP sensor protein. cGMP and Ca2+ dynamics were visualized in individual CGNs in primary cultures prepared from 7-day-old cGMP sensor mice. DEA/NO increased the intracellular cGMP concentration and augmented glutamate-induced Ca2+ transients. These effects of DEA/NO were absent in CGNs isolated from knockout mice lacking NO-sensitive guanylyl cyclase. Furthermore, application of the cGMP analogues 8-Br-cGMP and 8-pCPT-cGMP, which activate cGMP effector proteins such as cyclic nucleotide-gated cation channels and cGMP-dependent protein kinases (cGKs), also potentiated glutamate-induced Ca2+ transients. Western blot analysis failed to detect cGK type I or II in our primary CGNs. The addition of phosphodiesterase (PDE) inhibitors during cGMP imaging showed that CGNs degrade cGMP mainly via Zaprinast-sensitive PDEs, most likely PDE5 and/or PDE10, but not via PDE1, 2, or 3. In sum, these data delineate a cGK-independent NO-cGMP signaling cascade that increases glutamate-induced Ca2+ signaling in CGNs. This cGMP–Ca2+ crosstalk likely affects neurotransmitter-stimulated functions of CGNs.

Relaxin and extracellular matrix remodeling: Mechanisms and signaling pathways

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Relaxin and fibrosis: Emerging targets, challenges, and future directions

“…Technologies around real-time biosensors for ERK activity (de la Cova et al, 2017) and in vivo monitoring of ERK activity at a single cell resolution (Mayr et al, 2018) are also being developed. cGMP sensor mice might be valuable for cGMP measurements and for screening in vivo (Paolillo et al, 2018). ELISA kits are also available that allow for the measurement of cGMP from cell lysates, tissue homogenates, plasma or urine.…”

Hippocampal AMPA‐ and NMDA‐induced cGMP signals are mainly generated by NO‐GC2 and are under tight control by PDEs 1 and 2

“…Live cell fluorescent imaging of cGMP emerged as a method to analyse cGMP signals in a diversity of cell types including neurons and has recently been reviewed (Russwurm & Koesling, 2018). In the meantime, the Feil group reported on NO‐dependent cGMP in cerebellar granule cells and brain slices (Paolillo et al, 2018; Peters et al, 2018). Furthermore, the Vincent group analysed NMDA‐mediated stimulation of PDE1 activity in NO‐prestimulated striatal, cortical and hippocampal slices (Betolngar et al, 2019).…”