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Experimental design and recording setup. A local field potential (LFP) electrode, a glutamate biosensor and a glutamate null sensor were placed in the 3 rd -4 th cortical layers (left). After a 10-min baseline period in artificial cerebrospinal fluid (aCSF), slices were exposed to either hypo-osmotic medium (HM60) alone or HM60 supplemented with DCPIB, or TGN-020 + Bumetanide (TGN + Bum), or MK-801 + <t>CNQX</t> for 30 min. Transient hypoxia (2.5 min) elicited a spreading depolarization (SD), which was followed by a 5 min recovery period (right).
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Experimental design and recording setup. A local field potential (LFP) electrode, a glutamate biosensor and a glutamate null sensor were placed in the 3 rd -4 th cortical layers (left). After a 10-min baseline period in artificial cerebrospinal fluid (aCSF), slices were exposed to either hypo-osmotic medium (HM60) alone or HM60 supplemented with DCPIB, or TGN-020 + Bumetanide (TGN + Bum), or MK-801 + <t>CNQX</t> for 30 min. Transient hypoxia (2.5 min) elicited a spreading depolarization (SD), which was followed by a 5 min recovery period (right).
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Experimental design and recording setup. A local field potential (LFP) electrode, a glutamate biosensor and a glutamate null sensor were placed in the 3 rd -4 th cortical layers (left). After a 10-min baseline period in artificial cerebrospinal fluid (aCSF), slices were exposed to either hypo-osmotic medium (HM60) alone or HM60 supplemented with DCPIB, or TGN-020 + Bumetanide (TGN + Bum), or MK-801 + CNQX for 30 min. Transient hypoxia (2.5 min) elicited a spreading depolarization (SD), which was followed by a 5 min recovery period (right).

Journal: Frontiers in Cellular Neuroscience

Article Title: Glutamate load fosters spreading depolarization under osmotic stress in brain slices

doi: 10.3389/fncel.2025.1722194

Figure Lengend Snippet: Experimental design and recording setup. A local field potential (LFP) electrode, a glutamate biosensor and a glutamate null sensor were placed in the 3 rd -4 th cortical layers (left). After a 10-min baseline period in artificial cerebrospinal fluid (aCSF), slices were exposed to either hypo-osmotic medium (HM60) alone or HM60 supplemented with DCPIB, or TGN-020 + Bumetanide (TGN + Bum), or MK-801 + CNQX for 30 min. Transient hypoxia (2.5 min) elicited a spreading depolarization (SD), which was followed by a 5 min recovery period (right).

Article Snippet: Finally, NMDA receptors were blocked by the non-competitive NMDA receptor antagonist MK801 ( ; Tocris; 100 μM), co-applied with the AMPA/kainate receptor antagonist CNQX ( ; Tocris; 20 μM; n = 8; ).

Techniques:

Mechanistic summary of swelling-induced glutamate release and neuronal receptor activation during spreading depolarization. Under physiological conditions, astrocytes maintain extracellular glutamate and osmotic homeostasis via aquaporin-4 channels (AQP4), Na + -K + -2Cl − cotransporter 1 (NKCC1) and volume-regulated anion channel (VRAC), thereby preserving normal neuronal excitability. Osmotic stress promotes astrocyte swelling which activates VRAC to release glutamate, elevating extracellular glutamate and overstimulating neuronal NMDA N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors—this cascade fosters the occurrence of spreading depolarization. Pharmacological blockade—using DCPIB (VRAC blocker), Bumetanide (NKCC1 inhibitor), TGN-020 (AQP4 inhibitor), along with NMDA antagonist MK-801 and AMPA/kainate antagonist CNQX—attenuates astrocyte swelling, reduces glutamate accumulation and alleviates spreading depolarization. The figure was created in https://BioRender.com .

Journal: Frontiers in Cellular Neuroscience

Article Title: Glutamate load fosters spreading depolarization under osmotic stress in brain slices

doi: 10.3389/fncel.2025.1722194

Figure Lengend Snippet: Mechanistic summary of swelling-induced glutamate release and neuronal receptor activation during spreading depolarization. Under physiological conditions, astrocytes maintain extracellular glutamate and osmotic homeostasis via aquaporin-4 channels (AQP4), Na + -K + -2Cl − cotransporter 1 (NKCC1) and volume-regulated anion channel (VRAC), thereby preserving normal neuronal excitability. Osmotic stress promotes astrocyte swelling which activates VRAC to release glutamate, elevating extracellular glutamate and overstimulating neuronal NMDA N-methyl-D-aspartate (NMDA), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and kainate receptors—this cascade fosters the occurrence of spreading depolarization. Pharmacological blockade—using DCPIB (VRAC blocker), Bumetanide (NKCC1 inhibitor), TGN-020 (AQP4 inhibitor), along with NMDA antagonist MK-801 and AMPA/kainate antagonist CNQX—attenuates astrocyte swelling, reduces glutamate accumulation and alleviates spreading depolarization. The figure was created in https://BioRender.com .

Article Snippet: Finally, NMDA receptors were blocked by the non-competitive NMDA receptor antagonist MK801 ( ; Tocris; 100 μM), co-applied with the AMPA/kainate receptor antagonist CNQX ( ; Tocris; 20 μM; n = 8; ).

Techniques: Activation Assay, Preserving