Kirov, S. A. | bioRxiv Science

bioRxiv2024

Spreading depolarization causes reversible neuronal mitochondria fragmentation and swelling in healthy, normally perfused neocortex.

Mitochondrial function is tightly linked to their morphology, and fragmentation of dendritic mitochondria during noxious conditions suggests loss of function. In the normoxic cortex, spreading depolarization (SD) is a phenomenon underlying migraine aura. It is unknown whether mitochondria structure is affected by normoxic SD. In vivo two-photon imaging followed by quantitative serial section electron microscopy (ssEM) was used to monitor dendritic mitochondria in the normoxic cortex of urethane-anesthetized mature male and female mice during and after SD initiated by focal KCl microinjection. Structural dynamics of dendrites and their mitochondria were visualized by transfecting excitatory, glutamatergic neurons of the somatosensory cortex with bicistronic AAV, which induced tdTomoto labeling in neuronal cytoplasm and mitochondria labeling with roGFP. Normoxic SD triggered a rapid fragmentation of dendritic mitochondria alongside dendritic beading, both reversible; however, mitochondria took significantly longer to recover. Several rounds of SD resulted in transient mitochondrial fragmentation and dendritic beading without accumulating injury, as both recovered. SsEM corroborated normoxic SD-elicited dendritic and mitochondrial swelling and transformation of the filamentous mitochondrial network into shorter, swollen tubular and globular structures. Our results revealed normoxic SD-induced disruption of the dendritic mitochondrial structure that might impact mitochondrial bioenergetics during migraine with aura.

neuroscience↗

bioRxiv2022

Novel mechanism of hypoxic neuronal injury mediated by non-excitatory amino acids and astroglial swelling

Bleeding into cerebral parenchyma during hemorrhagic stroke or head trauma leads to ischemia and release of plasmatic content, including amino acids (AA). Although excitotoxic AA have been extensively studied, little is known about non-excitatory AA during hypoxic injury. Hypoxia-induced synaptic depression becomes irreversible after adding non-excitatory AA to hippocampal slices, alongside their intracellular accumulation and increased tissue electrical resistance. A combination of four non-excitatory AA (L-alanine, glycine, L-glutamine, L-serine: AGQS) at plasmatic concentrations was applied to brain slices from transgenic mice expressing EGFP in pyramidal neurons or astrocytes during normoxia or hypoxia. Two-photon imaging, changes in light transmittance (LT), and electrophysiological field recordings followed by electron microscopy in hippocampal CA1 st. radiatum were used to monitor synaptic function concurrently with cellular swelling and injury. During normoxia, AGQS-induced increase in LT was due to astroglial but not neuronal swelling. Fast LT raise during hypoxia and AGQS manifested neuronal and astroglial swelling accompanied by a permanent loss of synaptic transmission and irreversible dendritic beading, signifying acute tissue damage. Neuronal injury was not triggered by spreading depolarization which did not occur in our experiments. Hypoxia without AGQS did not cause cell swelling, leaving dendrites intact. Inhibition of NMDA receptors prevented neuronal damage and irreversible loss of synaptic function. Deleterious effects of AGQS during hypoxia were prevented by alanine-serine-cysteine transporters (ASCT2) and volume-regulated anion channels (VRAC) blockers. Our findings suggest that swelling induced by intracellular accumulation of non-excitatory AA and release of excitotoxins through antiporters and VRAC may exacerbate the hypoxia-induced neuronal injury. Significance StatementLittle is known if non-excitatory amino acids (AA) contribute to cellular injury when released during bleeding, as in hemorrhagic stroke and head trauma. Alanine, glycine, glutamine, and serine are one of the most abundant in plasma. Remarkably, during hypoxia, these non-excitatory AA caused severe neuronal and astroglial swelling and irreversible dendritic injury alongside a permanent loss of synaptic function. Activation of NMDA receptors was implicated in the onset of damage. Experimental evidence pointed to the involvement of alanine-serine-cysteine transporter 2 (ASCT2) and volume-regulated anion channels (VRAC) as molecular mechanisms underlying AA-induced damage during hypoxia. A detailed understanding of how brain injury evolves with non-excitatory AA during hypoxia will help design brain recovery treatments in neurological conditions involving bleeding.

neuroscience↗