Cx3cr1-BAC-CRE-mediated knockout of toll-like receptor 4 alters mouse communicative behaviors, microglial morphology, and engulfment of synaptic material

Bankowski, I. M.; Bishnoi, I. R.; Shin, E. E.; Cashin, M. J.; Möhrle, D.; Norris, H. A.; Barry, D. T.; Slamin, M. M.; Bordt, E. A.

doi:10.64898/2026.09.17.752150

bioRxiv · 10.64898/2026.09.17.752150

Cx3cr1-BAC-CRE-mediated knockout of toll-like receptor 4 alters mouse communicative behaviors, microglial morphology, and engulfment of synaptic material

Bankowski, I. M.·Bishnoi, I. R.·Shin, E. E.·Cashin, M. J.·Möhrle, D.·Norris, H. A.·Barry, D. T.·Slamin, M. M.·Bordt, E. A.

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Abstract

Microglia, the tissue resident immune cells of the central nervous system, are critical regulators of postnatal neural circuit refinement. Innate immune receptors on microglia such as toll-like receptor 4 (TLR4) are best characterized in the context of inflammation, however, endogenous TLR4 ligands are generated during typical developmental processes. Here, we investigated whether TLR4 signaling in CX3CR1-expressing myeloid cells contributes to microglial morphology, synaptic engulfment, and behavioral development in mice under basal conditions. TLR4 conditional knockout altered maternal separation-induced ultrasonic vocalizations without impacting social preference or anxiety-like behaviors. Conditional TLR4 knockout in CX3CR1-expressing myeloid cells markedly increased microglial ramification and cell volume within the paraventricular nucleus (PVN) of the hypothalamus. TLR4 conditional knockout also reduced microglial engulfment of vGlut2-positive presynaptic material, while overall excitatory synapse numbers remained unchanged. Together, these findings demonstrate that TLR4 signaling in CX3CR1-expressing myeloid cells contributes to microglial morphology, presynaptic engulfment, and neonatal communicative behavior under basal conditions.

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Bankowski, I. M., Bishnoi, I. R., Shin, E. E., Cashin, M. J., Möhrle, D., Norris, H. A., Barry, D. T., Slamin, M. M., Bordt, E. A.. 2026-09-23. Cx3cr1-BAC-CRE-mediated knockout of toll-like receptor 4 alters mouse communicative behaviors, microglial morphology, and engulfment of synaptic material. https://doi.org/10.64898/2026.09.17.752150

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