TNF-α and IFN-γ impair neural oscillations and induce neurodegeneration by microglial nitric oxide, metabolic and oxidative stress

BACKGROUND: The cytokine tumor necrosis factor-alpha (TNF-α) regulates inflammatory responses in infectious and neurodegenerative diseases and also affects neuronal function. The role of TNF-α in the activation of microglial cells (resident central nervous system macrophages), including the impact on neuronal survival, excitability, and synaptic transmission is incompletely defined, however. We explored the effects of chronic TNF-α exposure (72 h) on microglia and neurons in organotypic hippocampal slice cultures from male and female rats, i.e., postnatal cortex tissue lacking leukocyte invasion and adaptive immunity. METHODS: We applied gene expression analysis, biochemical assays, immunohistochemistry, electrophysiology by extracellular (local field potential) and intracellular (intrinsic membrane properties) recordings, and pharmacological ablation of the microglial cell population. We mainly focused on carbachol-induced neural network oscillations (brain waves) in the gamma frequency band (30-70 Hz) that underlie higher cognitive functions such as perception, attention, and memory. RESULTS: TNF-α induced microglial proliferation and upregulation of genes related to inflammation and oxidative stress such as Il6 (interleukin-6), Nos2 [inducible nitric oxide (NO) synthase, iNOS] and Sod2 (superoxide dismutase 2), which was accompanied by a decreased number of slices showing gamma oscillations in extracellular recordings. Notably, a fraction of slices presented neural bursting reflecting hyperexcitability in the tissue. Neuronal dysfunction was absent during acute TNF-α exposure (30 min). When paired with the lymphocyte cytokine interferon-gamma (IFN-γ), TNF-α induced an amplified neuroinflammation response dominated by bursting or loss of electrical activity. In intracellular recordings, neurons showed a brief burst of action potentials followed by slowing of spiking with pronounced afterhyperpolarization (switch from regular to burst firing behavior) during depolarizing current injection. Notably, the impairments could be attenuated by inhibition of iNOS and NADPH oxidase, glucose supplementation, microglial depletion or blockade of TNF receptor 1 (TNFR1) signaling with small molecule drugs, RIPA-56 and ICCB-19. CONCLUSIONS: Our data provide mechanistic insight into TNF-α- and IFN-γ-induced neuronal impairments mediated by microglial NO, metabolic and oxidative stress, and demonstrate functional neuroprotection by pharmacology. Our study extends the pathophysiological understanding of diseases such as sepsis, multiple sclerosis, Alzheimer's disease, depression and schizophrenia featuring activated microglia, infiltrating monocytes and T cells, and/or blood-brain barrier leakage.