The NKCC1 ion transporter contributes to the pathophysiology of common neurological disorders, but its function in microglia, the main inflammatory cells of the brain, has remained unclear to date. Therefore, we generated a novel transgenic mouse line in which microglial NKCC1 was deleted. We show that microglial NKCC1 shapes both baseline and reactive microglia morphology, process recruitment to the site of injury, and adaptation to changes in cellular volume in a cell-autonomous manner via regulating membrane conductance. In addition, microglial NKCC1 deficiency results in NLRP3 inflammasome priming and increased production of interleukin-1β (IL-1β), rendering microglia prone to exaggerated inflammatory responses. In line with this, central (intracortical) administration of the NKCC1 blocker, bumetanide, potentiated intracortical lipopolysaccharide (LPS)-induced cytokine levels. In contrast, systemic bumetanide application decreased inflammation in the brain. Microglial NKCC1 KO animals exposed to experimental stroke showed significantly increased brain injury, inflammation, cerebral edema, and, worse, neurological outcome. Thus, NKCC1 emerges as an important player in controlling microglial ion homeostasis and inflammatory responses through which microglia modulate brain injury. The contribution of microglia to central NKCC1 actions is likely to be relevant for common neurological disorders.

The NKCC1 ion transporter contributes to the pathophysiology of common neurological disorders, but its function in microglia, the main inflammatory cells of the brain, has not been studied to date. Therefore, we generated a novel transgenic mouse line in which microglial NKCC1 was deleted. We show that microglial NKCC1 shapes both baseline and reactive microglia morphology and inflammatory responses. As opposed to systemic NKCC1 blockade, which decreased intracortical lipopolysaccharide (LPS)-induced cytokine levels, neuroinflammation was potentiated by both intracortically administered bumetanide and in the absence of microglial NKCC1, suggesting a considerable role for microglia to influence central NKCC1 actions. Correspondingly, microglial NKCC1 KO animals exposed to experimental stroke showed significantly increased brain injury, inflammation, cerebral edema and worse neurological outcome. Thus, NKCC1 emerges as an important player to shape microglial responses and brain inflammation after CNS injury, which is likely to be relevant for common neurological disorders.

Microglia, the main immune cells of the central nervous system (CNS) have long been known for their remarkable sensitivity to tissue disturbance or injury, but its implications to the interpretation of results from ex vivo models of the CNS have remained largely unclear to date. To this end, we have followed the course of microglial phenotype changes and contribution to neuronal network organisation and functioning in acute brain slices prepared from mice, widely used to study the physiology of the brain from nanoscale events to complex circuits. We found that upon acute slice preparation, microglial cell bodies dislocate and migrate towards the surface of slices, alongside with rapidly progressing morphological changes and altered interactions with neurons. This is accompanied by gradual depolarization and downregulation of P2Y12 receptors, which are instrumental for microglia-neuron communication. Quantitative post-embedding immunofluorescent labelling reveals time-dependent increase in the number of excitatory and inhibitory synapses upon slice preparation in the cerebral cortex, which are markedly influenced by microglia. In line with this, the absence of microglia diminishes the incidence, amplitude and frequency of sharp wave-ripple activity in hippocampal slices. Collectively, our data suggest that microglia are not only inherent modulators of complex neuronal networks, but their specific actions on network reorganisation and functioning must be taken into account when learning lessons from ex vivo models of the CNS.