Forebrain microglia-associated synaptic alterations during obesity-related MASLD development

Metabolic dysfunction-associated steatotic liver disease (MASLD), considered as the hepatic manifestation of metabolic syndrome, is linked to cardiometabolic disease development. In addition to peripheral mechanisms, accumulating evidence points to changes in the brain as a factor in MASLD. In particular, we have demonstrated that excitatory signaling from the subfornical organ (SFO), a circumventricular nucleus located outside of the blood-brain-barrier, contributes to hepatic steatosis. While the mechanisms remain unclear, recent evidence indicates that microglia, the immune cell population within the brain, contribute to a number of diseases through alterations in synaptic contacts with neuron populations. Based on this, we hypothesized that SFO microglia contribute to MASLD development during obesity, and further, that this is related to changes in microglia-neuron synaptic interactions. To test this, male C57BL/6J mice were fed a high fat diet (HFD, 60% kcal fat) to induce hepatic steatosis or normal chow for 8 weeks. A chemogenetic strategy was then utilized to selectively inhibit microglial through SFO microinjection of a designer receptors exclusively activated by designer drugs inhibitory viral construct (AAV-CD68-hM4Gi-mCherry). Following surgical recovery, the pharmacological ligand clozapine-N-oxide (CNO; 3 mg/kg i.p., saline served as a control, n=7-11/group) was administered once daily over 3 days to inhibit SFO microglia. Selective inhibition of SFO microglia reduced liver lipid accumulation in mice fed a HFD (e.g. liver triglycerides: 2.7±0.3 vs 1.8±0.2 mM, HFD saline vs CNO, p<0.05) without altering body weight or food intake. Building upon this, we explored whether SFO microglia-neuronal synaptic alterations occurred during MASLD development. Male C57BL/6J mice fed a HFD or normal chow for 8 weeks were microinjected with a vector encoding green fluorescent protein driven under the glutamatergic neuron promoter calcium/calmodulin dependent kinase II (AAV-CAMKII-GFP) to label SFO excitatory neurons, which were then evaluated 3 weeks later using immunohistochemistry [microglia: ionized calcium-binding adapter molecule 1; CAMKII: GFP; inhibitory neurons: vesicular gamma-aminobutyric acid transporter (VGAT)]. Imaris software was used for analysis of synaptic interactions between cell types. 3D analysis throughout the SFO indicated that the number and surface area of SFO CAMKII neurons did not differ between groups (e.g. # CAMKII neurons: 135±16 vs 145±19, normal chow vs HFD, p>0.05). However, the total area of SFO microglia, reflecting microglial activation, was increased in HFD fed mice (75093±14206 vs 167712±29863 µm 2 , normal chow vs HFD, p<0.05). Interestingly, HFD fed mice demonstrated more than double the number of SFO CAMKII neurons in synaptic contact with microglia compared to control animals (41±4 vs 112±19, normal chow vs HFD, p<0.05). Moreover, the area of microglial synaptic coverage of SFO CAMKII neurons was also increased in HFD fed mice (64±7 vs 126±16 µm 2 , normal chow vs HFD, p<0.05). Surprisingly, increased microglial contact with SFO excitatory neurons was associated with reduced inhibitory synaptic innervation onto SFO CAMKII neurons (e.g. VGAT contact area: 160±15 vs 106±17 µm 2 ; VGAT contact area/CAMKII neuron: 22±3 vs 15±2%, normal chow vs HFD, p<0.05). Collectively, these findings indicate that: 1) SFO microglia activation contributes to MASLD development during obesity, and; 2) SFO microglia may alter excitatory/inhibitory neuronal balance by disrupting physical interactions between neuron populations. R01DK117007, R01HL141393, AHA932522 This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.