Extended Data Fig. 5: In vitro and in vivo microglial responses to gangliosides and cortical dysfunction in Hexb-deficient mice.
From: Microglia–neuron crosstalk through Hex–GM2–MGL2 maintains brain homeostasis
A-B) Cytokines and chemokine concentrations in supernatants from primary wildtype microglia cultured with the indicated amount of GM2, with/without EGTA or GalNAc. Data from 4 technical replicates. C) Fold changes of indicated cytokines normalized to the unstimulated condition (based on A and B) D) Schematic of in vivo experimental design. E-F) Cytokines/chemokine levels in brain lysates following ICV antibody injections. G) Quantitative PCR analysis of indicated target genes. H) Experimental setup. I) Representative traces (top) and current-clamp recording protocol (bottom). Note fewer action potentials (APs) and absence of voltage sag (arrow) in Hexb−/− compared to Hexb+/− mice. J) No difference in basic neuronal attributes between the genotypes (Hexb+/−: 4 animals, 11 neurons; Hexb−/−:4 animals, 11 neurons). K) Hexb−/− mice display reduced AP firing in response to depolarizing current injections across the entire range tested (left), along with an increase in rheobase, the amount of current required to elicit an AP (right, Hexb+/−-: 4 animals, 11 neurons; Hexb−/−: 4 animals, 11 neurons). L) Representative traces of individual APs. M) Hexb−/− mice show increased AP halfwidth, whereas AP amplitude and threshold are similar between the genotypes (Hexb+/−: 4 animals, 10 neurons; Hexb−/−:4 animals, 10/8/8 neurons respectively) N) Voltage sag during hyperpolarization (arrow in panel b) is reduced in Hexb−/− mice (Hexb+/−: 4 animals, 11 neurons; Hexb−/−:4 animals, 11 neurons). O) Voltage-clamp recordings of spontaneous excitatory postsynaptic currents (sEPSCs) show. robust reduction of sEPSC frequency in Hexb−/− mice. sEPSC amplitude is similar in both genotypes (right, Hexb+/−-: 4 animals, 9 neurons; Hexb−/−:4 animals, 8 neurons). Data shown as mean ± s.e.m. Statistical analyses: Two-way ANOVA followed by Sidak’s multiple comparison test (A-B,E-G); One-way ANOVA followed by Dunnett’s test (C); two-tailed Student’s t-test (I-K, M-O); two tailed Mann-Whitney (K). For I-O, dots correspond to individual neurons. Illustrations in d and h were created using BioRender (https://biorender.com).
