Human neutrophils communicate remotely via calcium-dependent glutamate-induced glutamate release

Summary Neutrophils are white blood cells that are critical to acute inflammatory and adaptive immune responses. Their swarming-pattern behavior is controlled by multiple cellular cascades involving calcium-dependent release of various signaling molecules. Previous studies have reported that neutrophils express glutamate receptors and can release glutamate but evidence of direct neutrophil-neutrophil communication has been elusive. Here, we hold semi-suspended cultured human neutrophils in patch-clamp whole-cell mode to find that calcium mobilization induced by stimulating one neutrophil can trigger an N-methyl-D-aspartate (NMDA) receptor-driven membrane current and calcium signal in neighboring neutrophils. We employ an enzymatic-based imaging assay to image, in real time, glutamate release from neutrophils induced by glutamate released from their neighbors. These observations provide direct evidence for a positive-feedback inter-neutrophil communication that could contribute to mechanisms regulating communal neutrophil behavior

Volume-transmitted GABA waves pace epileptiform rhythms in the hippocampal network

Mechanisms that entrain and pace rhythmic epileptiform discharges remain debated. Traditionally, the quest to understand them has focused on interneuronal networks driven by synaptic GABAergic connections. However, synchronized interneuronal discharges could also trigger the transient elevations of extracellular GABA across the tissue volume, thus raising tonic conductance (Gtonic) of synaptic and extrasynaptic GABA receptors in multiple cells. Here, we monitor extracellular GABA in hippocampal slices using patch-clamp GABA "sniffer" and a novel optical GABA sensor, showing that periodic epileptiform discharges are preceded by transient, region-wide waves of extracellular GABA. Neural network simulations that incorporate volume-transmitted GABA signals point to a cycle of GABA-driven network inhibition and disinhibition underpinning this relationship. We test and validate this hypothesis using simultaneous patch-clamp recordings from multiple neurons and selective optogenetic stimulation of fast-spiking interneurons. Critically, reducing GABA uptake in order to decelerate extracellular GABA fluctuations-without affecting synaptic GABAergic transmission or resting GABA levels-slows down rhythmic activity. Our findings thus unveil a key role of extrasynaptic, volume-transmitted GABA in pacing regenerative rhythmic activity in brain networks

Human neutrophils communicate remotely via calcium-dependent glutamate-induced glutamate release

Summary: Neutrophils are white blood cells that are critical to acute inflammatory and adaptive immune responses. Their swarming-pattern behavior is controlled by multiple cellular cascades involving calcium-dependent release of various signaling molecules. Previous studies have reported that neutrophils express glutamate receptors and can release glutamate but evidence of direct neutrophil-neutrophil communication has been elusive. Here, we hold semi-suspended cultured human neutrophils in patch-clamp whole-cell mode to find that calcium mobilization induced by stimulating one neutrophil can trigger an N-methyl-D-aspartate (NMDA) receptor-driven membrane current and calcium signal in neighboring neutrophils. We employ an enzymatic-based imaging assay to image, in real time, glutamate release from neutrophils induced by glutamate released from their neighbors. These observations provide direct evidence for a positive-feedback inter-neutrophil communication that could contribute to mechanisms regulating communal neutrophil behavior

Volume-transmitted GABA waves pace epileptiform rhythms in the hippocampal network.

Mechanisms that entrain and pace rhythmic epileptiform discharges remain debated. Traditionally, the quest to understand them has focused on interneuronal networks driven by synaptic GABAergic connections. However, synchronized interneuronal discharges could also trigger the transient elevations of extracellular GABA across the tissue volume, thus raising tonic conductance (Gtonic) of synaptic and extrasynaptic GABA receptors in multiple cells. Here, we monitor extracellular GABA in hippocampal slices using patch-clamp GABA sniffer and a novel optical GABA sensor, showing that periodic epileptiform discharges are preceded by transient, region-wide waves of extracellular GABA. Neural network simulations that incorporate volume-transmitted GABA signals point to a cycle of GABA-driven network inhibition and disinhibition underpinning this relationship. We test and validate this hypothesis using simultaneous patch-clamp recordings from multiple neurons and selective optogenetic stimulation of fast-spiking interneurons. Critically, reducing GABA uptake in order to decelerate extracellular GABA fluctuations-without affecting synaptic GABAergic transmission or resting GABA levels-slows down rhythmic activity. Our findings thus unveil a key role of extrasynaptic, volume-transmitted GABA in pacing regenerative rhythmic activity in brain networks

Volume-transmitted GABA waves pace epileptiform rhythms in the hippocampal network

Funding Information: This work was supported by Wellcome Principal Fellowship (212251/Z/18/Z and 212285/Z/18/Z), Wellcome Collaborative Award (UNS120639 - 223131/Z/21/Z), Epilepsy Research UK (F1901), MRC (MR/W019752/1, MR/V013556/1, and MR/V034758/1), NC3Rs (NC/X001067/1), and ERC Advanced Grant (323113). Optimization and parallelization of ARACHNE algorithms for extended neural network simulations was provided by AMC Bridge (Waltham, MA) and web security by Cyber Curio (Berkhamsted, UK). L.P.S. and D.A.R. narrated the study. I.P. and V.M. designed and carried out electrophysiological and optogenetic studies. N.C. O.K. and T.P.J. designed, carried out, and analyzed iGABASnFR2 imaging experiments. S.S. carried out sniffer-patch experiments. O.T. carried out control-probing K+ imaging tests. L.P.S. designed and carried out network modeling studies and data analyses. J.S.M. L.L.L. J.P.H. and I.K. supplied optical GABA sensors and related protocols. The development of iGABASnFR2 was conducted under the aegis of the HHMI Janelia GENIE Project at Janelia Research Campus. D.M.K. and M.C.W. designed optogenetic experiments. D.A.R. designed selected experiments and simulations, carried out selected analyses, and wrote the manuscript draft, with contributions from V.M. L.P.S. and all other authors. The authors declare no competing interests. We support inclusive, diverse, and equitable conduct of research. Publisher Copyright: © 2023 The Author(s)Peer reviewedPublisher PD