A wake-like state in vitro induced by transmembrane TNF/soluble TNF receptor reverse signaling.

Tumor necrosis factor alpha (TNF) has sleep regulatory and brain development roles. TNF promotes sleep in vivo and in vitro while TNF inhibition diminishes sleep. Transmembrane (tm) TNF and the tmTNF receptors (Rs), are cleaved by tumor necrosis factor alpha convertase to produce soluble (s) TNF and sTNFRs. Reverse signaling occurs in cells expressing tmTNF upon sTNFR binding. sTNFR administration in vivo inhibits sleep, thus we hypothesized that a wake-like state in vitro would be induced by sTNFR-tmTNF reverse signaling. Somatosensory cortical neuron/glia co-cultures derived from male and female mice lacking both TNFRs (TNFRKO), or lacking TNF (TNFKO) and wildtype (WT) mice were plated onto six-well multi-electrode arrays. Daily one-hour electrophysiological recordings were taken on culture days 4 through 14. sTNFR1 (0.0, 0.3, 3, 30, 60, and 120 ng/µL) was administered on day 14. A final one-hour recording was taken on day 15. Four measures were characterized that are also used to define sleep in vivo: action potentials (APs), burstiness index (BI), synchronization of electrical activity (SYN), and slow wave power (SWP; 0.25-3.75 Hz). Development rates of these emergent electrophysiological properties increased in cells from mice lacking TNF or both TNFRs compared to cells from WT mice. Decreased SWP, after the three lowest doses (0.3, 3 and 30 ng/µL) of the sTNFR1, indicate a wake-like state in cells from TNFRKO mice. A wake-like state was also induced after 3 ng/µl sTNFR1 treatment in cells from TNFKO mice, which express the TNFR1 ligand, lymphotoxin alpha. Cells from WT mice showed no treatment effects. Results are consistent with prior studies demonstrating involvement of TNF in brain development, TNF reverse signaling, and sleep regulation in vivo. Further, the current demonstration of sTNFR1 induction of a wake-like state in vitro is consistent with the idea that small neuronal/glial circuits manifest sleep- and wake-like states analogous to those occurring in vivo. Finally, that sTNF forward signaling enhances sleep while sTNFR1 reverse signaling enhances a wake-like state is consistent with other sTNF/tmTNF/sTNFR1 brain actions having opposing activities.

The neuron-specific interleukin-1 receptor accessory protein alters emergent network state properties in Vitro.

Small in vitro neuronal/glial networks exhibit sleep-like states. Sleep regulatory substance interleukin-1ß (IL1) signals via its type I receptor and a receptor accessory protein (AcP). AcP has a neuron-specific isoform called AcPb. After sleep deprivation, AcPb, but not AcP, upregulates in brain, and mice lacking AcPb lack sleep rebound. Herein we used action potentials (APs), AP burstiness, synchronization of electrical activity (SYN), and delta wave (0.5-3.75 Hz) power to characterize cortical culture network state. Homologous parameters are used in vivo to characterize sleep. Cortical cells from 1-2-day-old pups from AcP knockout (KO, lacking both AcP and AcPb), AcPb KO (lacking only AcPb), and wild type (WT) mice were cultured separately on multi-electrode arrays. Recordings of spontaneous activity were taken each day during days 4-14 in vitro. In addition, cultures were treated with IL1, or in separate experiments, stimulated electrically to determine evoked response potentials (ERPs). In AcP KO cells, the maturation of network properties accelerated compared to those from cells lacking only AcPb. In contrast, the lack of AcPb delayed spontaneous network emergence of sleep-linked properties. The addition of IL1 enhanced delta wave power in WT cells but not in AcP KO or AcPb KO cells. The ontology of electrically-induced ERPs was delayed in AcP KO cells. We conclude IL1 signaling has a critical role in the emergence of sleep-linked network behavior with AcP playing a dominant role in the slowing of development while AcPb enhances development rates of sleep-linked emergent network properties.