Role of IL-1b in Inflammation-Mediated Neural Circuit Formation Defects In Vivo

Background: Neuroinflammation initiated by maternal infection during fetal development has been strongly implicated in the etiology of neurodevelopmental disorders, including autism spectrum disorders (ASD) and schizophrenia. Little is yet known about the precise mechanisms that contribute to neurodevelopmental defects caused by inflammation.

Objectives: Using the zebrafish larva as a genetically tractable model, we sought to study the effects of inflammatory insults on neurodevelopmental programs in the retinotectal system in vivo.

Methods: Zygotic microinjection of Brn3c:Gal4 and UAS:mYFP plasmids allowed for sparse labeling of retinal ganglion cell (RGC) axons. At 3 days post-fertilization (dpf), when RGCs enter the optic tectum, we treated the larvae with bacterial lipopolysaccharide (LPS) to induce inflammation. We imaged the cells in vivo by 2-photon microscopy and reconstructed the arbors for morphometric analysis. The experimenter was blinded to the condition until the analysis was completed.

Results: We show that treatment of zebrafish larvae with LPS causes an increase in RGC branching dynamics immediately after the inflammatory insult (145.4% increase in number of branches added, P = 0.0068, 119.7% increase in number of branches lost, P = 0.033, 2-way ANOVA, n = 10 LPS treated, n = 9 untreated). We also saw an increase in overall size and branch numbers in LPS treated animals over the following several days (121.9% increase in total arbor length at 6 dpf, P = 0.0075, 158% increase in total number of branches, P = 0.0077, 2-way ANOVA, n = 18 LPS treated, n = 18 untreated). mRNA levels of the pro-inflammatory cytokine IL-1b levels are increased 6.8-fold following LPS treatment (P = 0.012, K-W test, n = 7), and morpholino oligonucleotide (MO) knock-down of this cytokine negates the effects of LPS treatment. Delay of specification of the myeloid lineage, which includes microglia, by MO knockdown of the PU.1 transcription factor, eliminates the immediate effects on RGC branching dynamics (90.3% of number of branches added, P = 0.6445, 112.1% of number of branches lost, P = 0.345, 2-way ANOVA, n = 8 LPS treated, n = 7 untreated), indicating a role for microglia in the mechanisms that mediate inflammation-induced neuronal defects.

Conclusions: We have shown that inflammation during neuronal development causes immediate changes in branching dynamics and long-term morphological defects in arborization. IL-1b is a critical cytokine in this process and mediates the effects of inflammatory stimulus on neuronal growth. Microglia are also involved in mediating the immediate effects of LPS treatment on cellular dynamics. Zebrafish larvae can be genetically manipulated in large numbers, permitting rapid candidate signal screening in vivo. Our findings will inform translational studies that can help us better understand and ultimately may prevent the occurrence of neurodevelopmental disorders such as autism.