Customer Article | Neuron | Neural Code of Social Avoidance: Hai-Tao Wu’s Team Reveals a New Mechanism of Dual Anterior Insula–Prefrontal Cortex Circuit Dialogue

Part 05 | Role of the AIV → PrL^OXTR Circuit in Modulating Social Novelty Preference

• Control mice exhibited robust Ca²⁺ responses when investigating novel conspecifics, whereas SUS mice showed markedly reduced responses (Figures 5E–G).
• Immunostaining confirmed that ~90% of GCaMP6s-positive neurons co-expressed OXTR (Figures 5C–D).
• Optogenetic activation of this pathway restored social novelty preference in SUS mice without affecting baseline social behavior (Figures 5H–M).
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Figure 5. OXTR signaling in the AIV^CaMKIIα → PrL circuit regulates social novelty preference
 

Part 06 | Oxytocinergic Input from PVN to PrL and Its Modulation of the AIV^CaMKIIα → PrL^OXTR Circuit

To identify the source of oxytocinergic inputs to PrL^OXTR neurons and their regulatory effect on the AIV→PrL pathway, retrograde monosynaptic tracing was performed in Oxtr-Cre mice (Figure 6A). The results revealed that direct presynaptic inputs to PrL^OXTR neurons mainly originated from the paraventricular nucleus of the hypothalamus (PVN), with sparse labeling from the supraoptic nucleus (SON), and the majority of labeled PVN neurons were oxytocin-positive (Figures 6B–D).

In Oxtr-Cre mice, AIV was injected with AAV-ChR2-EGFP and PrL with Cre-dependent AAV-DIO-mCherry, labeling AIV axon terminals and PrL^OXTR neurons, respectively (Figures 6E–F). Acute slice recordings showed that TGOT, an oxytocin receptor agonist, enhanced light-evoked excitatory postsynaptic currents (oEPSCs) in the AIV→PrL pathway, whereas the antagonist atosiban inhibited these currents (Figures 6G–H). TGOT also increased the paired-pulse ratio (PPR), while atosiban decreased it (Figures 6I–J), suggesting that OXTR activation enhances presynaptic release probability and modulates short-term synaptic plasticity.

In vivo, bilateral microinjection of TGOT into PrL of SUS mice (Figures 6K–L) significantly increased exploration time toward novel mice (Figures 6M–N), confirming that direct activation of OXTR in PrL restores social novelty preference in SUS mice.

Figure 6. PVN-derived oxytocinergic input modulates synaptic efficacy of the AIV^CaMKIIα → PrL^OXTR circuit and regulates social behavior

 

Part 07 | Overactivated AID^CaMKIIα → FrA Circuit Mediates Lateral Inhibition of the AIV^CaMKIIα → PrL Circuit via PV Interneurons in SUS Mice

Based on local inhibitory mechanisms within the insula, the study hypothesized that overactivation of the AID→FrA pathway in SUS mice suppresses the AIV→PrL circuit via aIC local GABAergic neurons. Multiple approaches were used to test this hypothesis.

Using RV retrograde tracing, Viaat-Cre mice received bilateral PrL injections of RV-helper virus, followed three weeks later by injection of trans-synaptic rabies virus into AIV. Starter projection neurons were labeled in aIC (TagBFP+mCherry+), and their upstream GABAergic inhibitory inputs were specifically visualized using Cre-dependent EGFP, enabling precise mapping of this inhibitory circuit (Figures 7A–B).

Retrograde AAV2/R-CaMKIIα-ChR2 injection into FrA specifically labeled AID^CaMKIIα → FrA neurons. Optical stimulation of aIC evoked excitatory postsynaptic potentials (EPSPs) in aIC GABA interneurons, indicating functional input (Figure 7C). Light-evoked currents were blocked by 1 μM TTX and rescued by 20 mM 4-AP, confirming monosynaptic connectivity (Figures 7D–F). Among aIC GABA interneurons projecting to AIV^CaMKIIα → PrL neurons, 83% received input from AID^CaMKIIα → FrA neurons, while 17% were unresponsive, showing that AID^CaMKIIα → FrA activity can drive aIC GABA interneurons to inhibit AIV^CaMKIIα → PrL neurons.

In SUS mice, intrinsic excitability of aIC GABA interneurons controlling AIV→PrL was significantly increased (Figures 7G–H), and 93% exhibited fast-spiking (FS) phenotypes (Figures 7I–J), indicating that these neurons are predominantly PV-positive inhibitory interneurons.

To confirm this, PV-Cre mice received monosynaptic rabies virus injections, leading to ChR2-EGFP expression specifically in aIC PV interneurons controlling AIV^CaMKIIα → PrL neurons (Figures 7K). About 90% of EGFP-positive neurons co-localized with PV (Figures 7L–M). Optogenetic activation of these neurons induced inhibitory postsynaptic currents (IPSCs) in AIV^CaMKIIα → PrL projection neurons, which were fully blocked by GABA receptor antagonists, confirming their inhibitory function (Figures 7N–O).

Fiber photometry recordings revealed that Ca²⁺ responses of aIC PV interneurons were significantly enhanced during novel social exploration in SUS mice (Figures 7P–T), indicating that increased inhibitory drive leads to suppressed AIV^CaMKIIα → PrL activity. Optogenetic inhibition (eNpHR3.0) of the same neurons significantly restored social novelty preference (Figures 7W–X). In summary, aIC PV interneuron activity mediates lateral inhibition of the AIV^CaMKIIα → PrL circuit, causing social novelty preference deficits in SUS mice.

Figure 7. Overactivated aIC PV microcircuit mediates AID^CaMKIIα → FrA inhibition of the AIV^CaMKIIα → PrL circuit in SUS mice

Conclusion

Using a CES mouse model, the study identified two parallel and functionally opposing circuits within the anterior insular cortex (aIC): the AID → FrA circuit mediates social fear and avoidance, while the AIV → PrL circuit regulates social novelty preference. Chronic stress overactivates the AID → FrA pathway, which exerts lateral inhibition on the AIV → PrL circuit via aIC PV interneurons, while also downregulating OXTR signaling in the PrL, collectively leading to social avoidance and deficits in social novelty preference. Optogenetic, chemogenetic, or pharmacological activation of the AIV → PrL circuit or enhancement of OXTR signaling effectively reverses stress-induced social deficits.

This study provides a comprehensive mechanistic framework at the circuit, cellular, and molecular levels for how chronic stress causes social dysfunction and offers precise therapeutic targets for related psychiatric disorders.

Figure 8. Summary diagram


All viral tools used in this study are available from Brain Case Biotech.