Mapping insular cortex immune associated neuronal networks:
We employed advanced viral tracing and activity-dependent labeling methods to map the immune-processing neural network associated with the posterior insular cortex (pIC).
Using a dual viral injection strategy, we injected two types of AAV based tracers into the pIC of FosTRAP2 mice:
• A retrograde tracer to identify neural terminals projecting to the pIC.
• An anterograde tracer to label monosynaptic downstream projections from the pIC.
Following a recovery period, inflammation was induced using Zymosan-induced peritonitis, a well-characterized model that previously demonstrated immune memory in the pIC. During peak inflammation, 4-hydroxytamoxifen (4-OHT) was administered to label neurons activated during the immune response. Next, we performed tissue clearing and analysis using the iDisco protocol, allowing for high-resolution mapping of neural circuits. Fluorescent signals were used to identify both input and output pathways of the pIC. Whole-brain imaging quantified labeled neurons based on fluorescence and spherical morphology, providing an unbiased view of the immune-associated neural network.
We were able to create an activity-dependent map of the pIC across the entire brain, largely in accordance with existing literature on pIC connectivity. Moreover, our further investigation into these neural circuits uncovered recruitment or suppression of specific pathways during peripheral inflammation. This additional layer of insight could set the stage for new neural targets, including the olfactory system and oxytocin-expressing neurons as new and unexpected candidates for intervention and potential promising candidates for neuromodulation in the context of immune-related disorders.
Targeted electrical stimulation of the insular cortex in humans:
Patients undergoing invasive recordings for seizure localization (stereo-EEG) were invited to participate in an experiment testing the hypothesis that targeted electrical stimulation of specific brain regions can modulate immune responses and brain connectivity. Ethical considerations and informed consent processes were strictly adhered to, ensuring voluntary participation and minimizing additional risks.
Focusing the InsCtx, Direct Current Stimulation (DCS) was applied to predetermined areas of the insular cortex of 13 epilepsy patients via stereo-EEG electrodes. Stimulation parameters were carefully calibrated to ensure safety while achieving effective modulation of neural activity. Blood samples were collected before and after stimulation to assess changes in key immune markers, such as cytokines, immune cell profiles, and inflammatory mediators. Functional brain network changes induced by DCS were quantified using stereo-EEG recordings and advanced connectivity analysis techniques. Stimulation of non-target brain regions or sham stimulation were included as control conditions.
Analysis of preliminary data collected revealed a change in power correlation values across brain regions, time, and frequency bands subsequent to intracranial insula stimulation. Furthermore, these changes were unique to individual patients and stimulation, revealing bidirectional changes in overall regional brain activity as a result of insula stimulation. We are still analyzing the additional patients to gain statistical power. Nevertheless, as we estimated, we may not find significant correlations under these conditions since the patients have no immune challenge. Nevertheless, the fallback position of this study is estimating safety of these procedures for further therapeutic development.
