scholarly journals Src family kinases activity is required for transmitting purinergic P2X7 receptor signaling in cortical spreading depression and neuroinflammation

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Lingdi Nie ◽  
Dongqing Ma ◽  
John P. Quinn ◽  
Minyan Wang
Keyword(s):  
Gene Expression ◽  
Mouse Brain ◽  
P2x7 Receptor ◽  
Cortical Spreading Depression ◽  
Spreading Depression ◽  
Glutamate Release ◽  
Brain Slices ◽  
Src Family Kinases ◽  
Receptor Signaling ◽  
Purinergic P2x7 Receptor

Abstract Background Purinergic P2X7 receptor plays an important role in migraine pathophysiology. Yet precise molecular mechanism underlying P2X7R signaling in migraine remains unclear. This study explores the hypothesis that P2X7 receptor transmits signaling to Src family kinases (SFKs) during cortical spreading depression (CSD) and neuroinflammation after CSD. Methods CSD was recorded using electrophysiology in rats and intrinsic optical imaging in mouse brain slices. Cortical IL-1β and TNFα mRNA levels were detected using qPCR. Glutamate release from mouse brain slices was detected using glutamate assay. Results The data showed that deactivation of SFKs by systemic injection of PP2 reduced cortical susceptibility to CSD in rats and CSD-induced IL-1β and TNF-α gene expression in rat ipsilateral cortices. Consistently, in mouse brain slices, inhibition of SFKs activity by saracatinib and P2X7 receptor by A740003 similarly reduced cortical susceptibility to CSD. When the interaction of P2X7 receptor and SFKs was disrupted by TAT-P2X7, a marked reduction of cortical susceptibility to CSD, IL-1β gene expression and glutamate release after CSD induction were observed in mouse brain slices. The reduced cortical susceptibility to CSD by TAT-P2X7 was restored by NMDA, and disrupting the Fyn-NMDA interaction using TAT-Fyn (39-57) but not disrupting Src-NMDA receptor interaction using TAT-Src (40-49) reduced cortical susceptibility to CSD. Furthermore, activation of P2X7 receptor by BzATP restored the TAT-Fyn (39-57)-reduced cortical susceptibility to CSD. Conclusion This study reveals that SFKs activity transmits P2X7 receptor signaling to facilitate CSD propagation via glutamatergic pathway and promote neuroinflammation, which is of particular relevance to migraine.

scholarly journals Src Family Kinases Activity is Required for Transmitting Purinergic P2X7 Receptor Signaling in Cortical Spreading Depression and Neuroinflammation

2021 ◽  
Author(s):  
Lingdi Nie ◽  
Dongqing Ma ◽  
John P Quinn ◽  
Minyan Wang
Keyword(s):  
Gene Expression ◽  
Mouse Brain ◽  
P2x7 Receptor ◽  
Cortical Spreading Depression ◽  
Spreading Depression ◽  
Glutamate Release ◽  
Brain Slices ◽  
Src Family Kinases ◽  
Purinergic P2x7 Receptor ◽  
Precise Molecular Mechanism

Abstract Background Purinergic P2X7 receptor plays a key role in migraine pathophysiology. Yet precise molecular mechanism underlying P2X7R signaling in migraine remains unclear. This study explores the hypothesis that P2X7 receptor transmits signaling to Src family kinases (SFKs) during cortical spreading depression (CSD) and CSD-induced neuroinflammation. Methods CSD was recorded using electrophysiology in rats, and intrinsic optical imaging in mouse brain slices. Cortical IL-1β and TNFα mRNA expression were detected using qPCR. Glutamate release in mouse brain slices was detected using glutamate assay. Results The data showed that systematic deactivation of SFKs by PP2 reduced cortical susceptibility to CSD in rats and CSD-induced IL-1b and TNF-a gene expression in rat ipsilateral cortices. Consistently, in mouse brain slices, inhibition of SFKs activity by saracatinib and P2X7 receptor by A740003 similarly reduced cortical susceptibility to CSD. When the interaction of P2X7 receptor-SFKs was disrupted by TAT-P2X7, a marked reduction of cortical susceptibility to CSD, CSD-induced IL-1b gene expression and glutamate release were observed in mouse brain slices. The reduced cortical susceptibility to CSD by TAT-P2X7 was restored by NMDA and disrupting Fyn-NMDA interaction using TAT-Fyn (39-57), but not disrupting Src-NMDA receptor using TAT-Src (40-49), reduced cortical susceptibility to CSD. Furthermore, activation of P2X7 receptor by BzATP restored the TAT-Fyn (39-57)-reduced cortical susceptibility to CSD. Conclusion This study reveals that SFKs activity mediates P2X7 receptor pore formation facilitating CSD propagation, CSD-induced neuroinflammation and glutamate release, of particular relevance to migraine.

scholarly journals TRPA1-Mediated Src Family Kinases Activity Facilitates Cortical Spreading Depression Susceptibility and Trigeminovascular System Sensitization

2021 ◽  
Vol 22 (22) ◽  
pp. 12273
Author(s):  
Lingdi Nie ◽  
Liwen Jiang ◽  
John P. Quinn ◽  
Blair D. Grubb ◽  
Minyan Wang
Keyword(s):  
Mouse Brain ◽  
Cortical Spreading Depression ◽  
Spreading Depression ◽  
Quantitative Polymerase Chain Reaction ◽  
Brain Slices ◽  
Src Family Kinases ◽  
Enzyme Linked Immunosorbent Assay ◽  
Trigeminovascular System ◽  
Cgrp Release ◽  
Migraine Pathogenesis

Transient receptor potential ankyrin 1 (TRPA1) plays a role in migraine and is proposed as a promising target for migraine therapy. However, TRPA1-induced signaling in migraine pathogenesis is poorly understood. In this study, we explored the hypothesis that Src family kinases (SFKs) transmit TRPA1 signaling in regulating cortical spreading depression (CSD), calcitonin gene-related peptide (CGRP) release and neuroinflammation. CSD was monitored in mouse brain slices via intrinsic optical imaging, and in rats using electrophysiology. CGRP level and IL-1β gene expression in mouse trigeminal ganglia (TG) was detected using Enzyme-linked Immunosorbent Assay and Quantitative Polymerase Chain Reaction respectively. The results showed a SFKs activator, pYEEI (EPQY(PO3H2)EEEIPIYL), reversed the reduced cortical susceptibility to CSD by an anti-TRPA1 antibody in mouse brain slices. Additionally, the increased cytosolic phosphorylated SFKs at Y416 induced by CSD in rat ipsilateral cerebral cortices was attenuated by pretreatment of the anti-TRPA1 antibody perfused into contralateral ventricles. In mouse TG, a SFKs inhibitor, saracatinib, restored the CGRP release and IL-1β mRNA level increased by a TRPA1 activator, umbellulone. Moreover, umbellulone promoted SFKs phosphorylation, which was reduced by a PKA inhibitor, PKI (14–22) Amide. These data reveal a novel mechanism of migraine pathogenesis by which TRPA1 transmits signaling to SFKs via PKA facilitating CSD susceptibility and trigeminovascular system sensitization.

scholarly journals Sarcoma Family Kinase-Dependent Pannexin-1 Activation after Cortical Spreading Depression Is Mediated by NR2A-Containing Receptors

2020 ◽  
Vol 21 (4) ◽  
pp. 1269 ◽  
Author(s):  
Fan Bu ◽  
Lingdi Nie ◽  
John P Quinn ◽  
Minyan Wang
Keyword(s):  
Mouse Brain ◽  
Protein Interactions ◽  
Cortical Spreading Depression ◽  
Spreading Depression ◽  
Regulatory Mechanism ◽  
Brain Slices ◽  
Pannexin 1 ◽  
Migraine Headaches ◽  
Aspartate Receptor ◽  
Channel Opening

Cortical spreading depression (CSD) is a propagating wave of depolarization followed by depression of cortical activity. CSD triggers neuroinflammation via the pannexin-1 (Panx1) channel opening, which may eventually cause migraine headaches. However, the regulatory mechanism of Panx1 is unknown. This study investigates whether sarcoma family kinases (SFK) are involved in transmitting CSD-induced Panx1 activation, which is mediated by the NR2A-containing N-methyl-D-aspartate receptor. CSD was induced by topical application of K+ to cerebral cortices of rats and mouse brain slices. SFK inhibitor, PP2, or NR2A–receptor antagonist, NVP–AAM077, was perfused into contralateral cerebral ventricles (i.c.v.) of rats prior to CSD induction. Co-immunoprecipitation and Western blot were used for detecting protein interactions, and histofluorescence for addressing Panx1 activation. The results demonstrated that PP2 attenuated CSD-induced Panx1 activation in rat ipsilateral cortices. Cortical susceptibility to CSD was reduced by PP2 in rats and by TAT-Panx308 that disrupts SFK–Panx1 interaction in mouse brain slices. Furthermore, CSD promoted activated SFK coupling with Panx1 in rat ipsilateral cortices. Moreover, inhibition of NR2A by NVP–AAM077 reduced elevation of ipsilateral SFK–Panx1 interaction, Panx1 activation induced by CSD and cortical susceptibility to CSD in rats. These data suggest NR2A-regulated, SFK-dependent Panx1 activity plays an important role in migraine aura pathogenesis.

scholarly journals Cortical Spreading Depression Can Be Triggered by Sensory Stimulation in Primed Wild Type Mouse Brain: a Mechanistic Insight to Migraine Aura Generation

2021 ◽  
Author(s):  
Sahin Hanalioglu ◽  
Aslihan Taskiran-Sag ◽  
Hulya Karatas ◽  
Buket Donmez-Demir ◽  
Sinem Yilmaz-Ozcan ◽  
...  
Keyword(s):  
Mouse Brain ◽  
Cortical Spreading Depression ◽  
Spreading Depression ◽  
Glutamate Release ◽  
Wild Type Mouse ◽  
Sensory Stimulation ◽  
Photic Stimulation ◽  
Normal Brain ◽  
Whisker Stimulation

Abstract Background: Unlike the spontaneously appearing aura in migraineurs, experimentally, cortical spreading depression (CSD), the neurophysiological correlate of aura is induced by non-physiological stimuli. Consequently, neural mechanisms involved in spontaneous CSD generation, which may provide insight how migraine starts in an otherwise healthy brain, remains largely unclear. We hypothesized that CSD can be physiologically induced by sensory stimulation in primed mouse brain. Methods: Cortex was made susceptible to CSD with partial inhibition of Na+/K+-ATPase by epidural application of a low dose of Na+/K+-ATPase blocker ouabain that does not induce repetitive CSDs or by knocking-down α2 subunit of Na+/K+-ATPase, which is crucial for K+ and glutamate re-uptake by astrocytes, with shRNA. Stimulation-induced CSDs and extracellular K+ changes were monitored in vivo electrophysiologically or with a K+-sensitive fluoroprobe (IPG-4). Results: After priming with ouabain, photic stimulation increased the CSD incidence compared with non-stimulated animals (44.0 vs. 4.9%, p<0.001). Whisker stimulation was less effective (14.9 vs. 2.4%, p=0.02). Knocking-down Na+/K+-ATPase (50% decrease in mRNA) lowered the CSD threshold in all mice tested but triggered stimulus-induced CSDs in 14.3% and 16.7% of mice with photic and whisker stimulation, respectively. Confirming Na+/K+-ATPase hypofunction, extracellular K+ significantly rose during stimulation after subthreshold ouabain or shRNA treatment unlike controls. In line with higher CSD susceptibility, K+ rise was more prominent after ouabain. To gain insight to preventive mechanisms reducing the incidence of stimulus-induced CSDs, we applied an A1-receptor (DPCPX) or GABA-A (bicuculine) antagonist over the occipital cortex, because adenosine formed during stimulation or inhibitory interneuron activity can reduce CSD susceptibility. DPCPX induced CSDs or CSD-like small-DC shifts during photic stimulation, whereas bicuculine was not effective. Conclusions: Our findings indicate that normal brain is well protected against CSD generation. For CSD to be ignited under physiological conditions, priming and predisposing factors are required as seen in migraine patients. Intense sensory stimulation has the potential to trigger a CSD when co-existing conditions can bring extracellular K+ and glutamate concentrations over threshold via reduced uptake of K+ and glutamate (e.g. inefficient fueling of α2-Na+/K+-ATPase due to reduced glycogen breakdown) or facilitated glutamate release (e.g. reduced presynaptic adenosinergic inhibition).

Astrocyte-mediated inflammation in cortical spreading depression

Cephalalgia ◽  
2017 ◽  
Vol 38 (4) ◽  
pp. 626-638 ◽  
Author(s):  
Amir Ghaemi ◽  
Leila Alizadeh ◽  
Shahnaz Babaei ◽  
Maryam Jafarian ◽  
Maryam Khaleghi Ghadiri ◽  
...  
Keyword(s):  
Inflammatory Markers ◽  
Cortical Spreading Depression ◽  
Spreading Depression ◽  
Ex Vivo ◽  
Brain Slices ◽  
Cerebrovascular Diseases ◽  
Protein Markers ◽  
Markers Of Inflammation ◽  
Reactive Astrocytosis

Background Cortical spreading depression (CSD) related diseases such as migraine, cerebrovascular diseases, and epilepsy have been associated with reactive astrocytosis, yet the mechanisms of these tissue changes remain unclear. CSD-induced inflammatory response has been proposed to play a role in some neurological disorders and thus may also contribute to reactive astrocytosis. Methods Using ex vivo brain slices and in vitro astrocytic cultures, we aimed to characterize CSD related changes in astrocytes and markers of inflammation by immunocyto- and immunohistochemistry. CSD was induced by application of KCl (3 mol/l) on neocortical tissues. The application of KCl was repeated weekly over the course of four weeks. Results CSD induced an increase in the mean number and volume of astrocytes in rat brain tissue when compared to controls, whereas no changes in neuronal numbers and volumes were seen. These cell-type specific changes, suggestive of reactive astrocytosis, were paralleled by an increased expression of protein markers indicative of astrocytes and neuroinflammation in ex vivo brain slices of animals undergoing CSD when compared to sham-treated controls. Cultured astrocytes showed an increased expression of the immune modulatory enzyme indoleamine 2,3-dioxygenase and an elevated expression of the pro-inflammatory markers, IL-6, IL-1β, and TNFα in addition to increased levels of toll like receptors (TLR3 and TLR4) and astrocytic markers after induction of CSD. Conclusion These findings indicate that CSD related reactive astrocytosis is linked to an upregulation of inflammatory markers. Targeting inflammation with already approved and available immunomodulatory treatments may thus represent a strategy to combat or ameliorate CSD-related disease.

scholarly journals A Glass Capillary Microelectrode Based on Capillarity and Its Application to the Detection of L-Glutamate Release from Mouse Brain Slices.

2003 ◽  
Vol 19 (1) ◽  
pp. 55-60 ◽  
Author(s):  
Kumiko NAKAJIMA ◽  
Takashi YAMAGIWA ◽  
Ayumi HIRANO ◽  
Masao SUGAWARA
Keyword(s):  
Mouse Brain ◽  
Glutamate Release ◽  
Brain Slices ◽  
Glass Capillary

Imaging mass spectrometry to visualize biomolecule distributions in mouse brain tissue following hemispheric cortical spreading depression

2012 ◽  
Vol 75 (16) ◽  
pp. 5027-5035 ◽  
Author(s):  
Emrys A. Jones ◽  
Reinald Shyti ◽  
René J.M. van Zeijl ◽  
Sandra H. van Heiningen ◽  
Michel D. Ferrari ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Brain Tissue ◽  
Mouse Brain ◽  
Cortical Spreading Depression ◽  
Spreading Depression ◽  
Imaging Mass Spectrometry

scholarly journals Regenerative Glutamate Release by Presynaptic NMDA Receptors Contributes to Spreading Depression

2013 ◽  
Vol 33 (10) ◽  
pp. 1582-1594 ◽  
Author(s):  
Ning Zhou ◽  
Ravi L Rungta ◽  
Aqsa Malik ◽  
Huili Han ◽  
Dong Chuan Wu ◽  
...  
Keyword(s):  
Nmda Receptors ◽  
Spreading Depression ◽  
Calcium Release ◽  
Glutamate Release ◽  
Brain Slices ◽  
Receptor Activation ◽  
Neuronal Communication ◽  
High K ◽  
Vesicular Exocytosis ◽  
Presynaptic Nmda Receptors

Spreading depression (SD) is a slowly propagating neuronal depolarization that underlies certain neurologic conditions. The wave-like pattern of its propagation suggests that SD arises from an unusual form of neuronal communication. We used enzyme-based glutamate electrodes to show that during SD induced by transiently raising extracellular K+ concentrations ([K+]o) in rat brain slices, there was a rapid increase in the extracellular glutamate concentration that required vesicular exocytosis but unlike fast synaptic transmission, still occurred when voltage-gated sodium and calcium channels (VGSC and VGCC) were blocked. Instead, presynaptic N-methyl-D-aspartate (NMDA) receptors (NMDARs) were activated during SD and could generate substantial glutamate release to support regenerative glutamate release and propagating waves when VGSCs and VGCCs were blocked. In calcium-free solutions, high [K+]o still triggered SD-like waves and glutamate efflux. Under such a condition, glutamate release was blocked by mitochondrial Na+/Ca2+ exchanger inhibitors that likely blocked calcium release from mitochondria secondary to NMDA-induced Na+ influx. Therefore presynaptic NMDA receptor activation is sufficient for triggering vesicular glutamate release during SD via both calcium entry and release from mitochondria by mitochondrial Na+/Ca2+ exchanger. Our observations suggest that presynaptic NMDARs contribute to a cycle of glutamate-induced glutamate release that mediate high [K+]o-triggered SD.

Cortical spreading depression-induced preconditioning in mouse neocortex is lamina specific

2013 ◽  
Vol 109 (12) ◽  
pp. 2923-2936 ◽  
Author(s):  
Helen M. Gniel ◽  
Rosemary L. Martin
Keyword(s):  
Cortical Spreading Depression ◽  
Spreading Depression ◽  
Pyramidal Neurons ◽  
Brain Slices ◽  
Glucose Deprivation ◽  
Time Frame ◽  
Degree Of Protection ◽  
Equilibrative Nucleoside Transporters ◽  
A Cell ◽  
Layer V

Cortical spreading depression (CSD) is able to confer neuroprotection when delivered at least 1 day in advance of an ischemic event. However, its ability to confer neuroprotection in a more immediate time frame has not previously been investigated. Here we have used mouse neocortical brain slices to study the effects of repeated episodes of CSD in layer V and layer II/III pyramidal neurons. In layer V, CSD evoked at 15-min intervals caused successively smaller membrane depolarizations and increases in intracellular calcium compared with the response to the first CSD. With an inter-CSD interval of 30 min this preconditioning effect was much less marked, indicating that preconditioning lasts between 15 and 30 min. A single episode of CSD also provided a degree of protection in oxygen-glucose deprivation (OGD) by significantly lengthening the time a cell could withstand OGD before anoxic depolarization occurred. In layer II/III pyramidal neurons no preconditioning by CSD on subsequent episodes of CSD was observed, demonstrating that the response of pyramidal neurons to repeated CSD is lamina specific. The A1 receptor antagonist 8-cyclopentyl theophylline (8-CPT) reduced the layer V preconditioning in a concentration-related manner. Inhibition of extracellular formation of adenosine by blocking ecto-5′-nucleotidase with α,β-methyleneadenosine 5′-diphosphate prevented preconditioning in most but not all cells. Block of equilibrative nucleoside transporters 1 and 2 with dipyramidole alone or in combination with 6-[(4-nitrobenzyl)thio]-9-β-d-ribofuranosylpurine also prevented preconditioning in some but not all cells. These data provide evidence that rapid preconditioning of one CSD by another is primarily mediated by adenosine.

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