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Neuron︱Zhejiang University’s Hailan Hu Team Analyzes the Neural Circuits Behind Social Hierarchy in Mice

Release time：2025-01-06 13:51:01

Social competition refers to interactions among animals aimed at establishing hierarchical status, a determination that has profound effects on behavioral patterns and overall health. The dorsomedial prefrontal cortex (dmPFC), a part of the brain's prefrontal cortex, has been shown in previous studies to play a critical role in encoding dominance status and achieving success in competition. In humans, primates, and rodents, dmPFC activity has been linked to an individual’s social rank and competitive outcomes. While some understanding of the dmPFC's role in social competition has been established, much remains unknown about how the dmPFC coordinates complex social behaviors through its downstream neural circuits, particularly behaviors associated with victory and defeat.

On December 10, 2024, Professor Hailan Hu's team from the Zhejiang University School of Brain Science and Brain Medicine and the Interdisciplinary Institute of Neuroscience and Technology published a research paper in Neuron titled "Deconstructing the Neural Circuit Underlying Social Hierarchy in Mice." The study revealed that the dmPFC regulates competitive behaviors by controlling distinct downstream pathways, shedding light on how the brain coordinates complex behaviors through top-down regulation. Specifically, layer 5 neurons in the dmPFC projecting to the dorsal raphe nucleus (DRN) and periaqueductal gray (PAG) promote social competition, while layer 2/3 neurons projecting to the anterior basolateral amygdala (aBLA) inhibit social competition.

Downstream Brain Regions of the dmPFC Associated with Victory or Defeat

Using adeno-associated virus (AAV)-based strategies, the researchers mapped the whole-brain projections of the dmPFC by labeling neurons with EGFP and axon terminals with synaptic protein-fused mRuby. The mapping revealed that dmPFC axon terminals are particularly dense in several brain regions, including the basolateral amygdala (BLA), caudate-putamen (CPu), nucleus accumbens (NAc), and mediodorsal thalamus (MDT).

To identify the downstream regions of the dmPFC involved in social competition, the researchers used the tube test to evaluate social hierarchy and competitive behavior in mice. During this test, behaviors such as push, resistance, and retreat were recorded. Mice that successfully pushed another mouse out of the tube and advanced were classified as dominant (higher rank), while mice that retreated were classified as subordinate (lower rank).

The researchers compared c-Fos immunoreactivity patterns in the brains of winners and losers following the tube test (Figure 1A). They found significant differences in c-Fos expression between the two groups (Figure 1B). Winning mice exhibited increased c-Fos expression in the dmPFC as well as in three downstream subcortical regions: MDT, DRN, and periaqueductal gray (PAG). In contrast, losing mice primarily showed increased c-Fos expression in the aBLA (Figures 1B and 1C).

Figure 1: Downstream Brain Regions of the dmPFC Associated with Victory or Defeat

Inhibiting Different dmPFC Downstream Circuits Alters Dominance Rank

To determine the functional roles of different dmPFC projections in regulating social hierarchy, AAV was used to bilaterally express eNpHR3.0 in the dmPFC, allowing the inhibition of these pathways. Optical fibers were implanted into various downstream brain regions (Figure 2) to selectively inhibit specific dmPFC projection terminals.

The results showed that optogenetic inhibition of the dmPFC-MDT pathway (Figures 2B–2D), the dmPFC-dorsomedial caudate-putamen (dmCPu), and the dmPFC-NAc pathways did not affect the dominance rank of mice in the tube test. Dominance rank reflects an individual’s position in the social hierarchy, with higher-ranked individuals often having priority access to food, mates, and territory, as well as greater influence in group decision-making.

In contrast, inhibition of the dmPFC-DRN pathway (Figures 2E–2H) or the dmPFC-PAG pathway (Figures 2I–2L) led to an increase in retreat behaviors (Figures 2H and 2L) and a reduction in dominance rank (Figures 2G and 2K). On the other hand, optogenetic inhibition of the dmPFC-aBLA pathway (Figures 2M–2P) reduced retreat behaviors (Figure 2P) and increased dominance rank (Figure 2O).

Figure 2: Inhibiting Different dmPFC Downstream Circuits Alters Dominance Rank

Activating Different dmPFC Downstream Circuits Alters Dominance Rank

An AAV virus encoding channelrhodopsin (ChR2) was injected into the right dmPFC, and optical fibers were implanted above the DRN, PAG, or aBLA (Figures 3A, 3E, and 3I) to activate the projection terminals of the dmPFC-DRN, dmPFC-PAG, and dmPFC-aBLA pathways. Activation of the dmPFC-DRN or dmPFC-PAG pathways promoted pushing behavior during the tube test and increased dominance rank (Figures 3B–3D and 3F–3H). In contrast, activation of the dmPFC-aBLA pathway increased retreat behaviors during the tube test and decreased dominance rank (Figures 3J–3L).

Notably, activation of the dmPFC-DRN, dmPFC-PAG, or dmPFC-aBLA pathways did not induce preference or aversion in a real-time place preference test. To further confirm the specificity of these projections, the researchers injected a retrograde virus (AAV2/2Retro-hSyn-Cre) into the DRN, PAG, or aBLA, and an AAV expressing Cre-inducible light-sensitive ChR2 into the dmPFC. This enabled specific activation of dmPFC neurons projecting to the DRN, PAG, or aBLA. Results showed that activating dmPFC neurons projecting to the DRN and PAG increased dominance rank, whereas activating those projecting to the aBLA decreased dominance rank.

These findings indicate that the behavioral changes induced by activating dmPFC neurons are consistent with the effects observed from activating their projection terminals.

Figure 3: Activating Different dmPFC Downstream Circuits Alters Dominance Rank

Regulating the aBLA Affects Rank in the Tube Test

Given the unique role of the dmPFC-aBLA pathway in regulating social competition, the authors further explored the function of this pathway by attempting to inhibit or activate the aBLA itself during the tube test (Figure 4).

When optogenetically inhibiting the aBLA (Figure 4A), seven out of nine mice exhibited more pushing behavior and fewer retreat behaviors, leading to an increase in dominance rank (Figures 4B–4D). In contrast, optogenetic activation of the aBLA decreased dominance rank in the tube test, with seven out of eight mice showing this effect (Figures 4E–4H).

Figure 4: Regulating the aBLA Affects Rank in the Tube Test

Activity Dynamics of Different dmPFC Projection Neuron Subgroups in the Tube Test

To examine the activity states of different dmPFC neuron subgroups during social competition, the authors used an optical fiber recording system to monitor their activity in real-time during tube test confrontations (Figures 5A and 5B).

AAV expressing Cre-inducible Ca2+ indicator GCaMP6s was injected into the right dmPFC, and a retrograde virus (AAV2/Retro-hSyn-Cre) was injected into the DRN, PAG, or aBLA. Following viral expression, optical fibers were implanted 200 micrometers above the injection site in the dmPFC to record Ca2+ signals (Figures 5C, 5G, and 5K).

Similar to the general pattern observed in the entire dmPFC, Ca2+ signals in dmPFC neurons projecting to the DRN significantly increased after pushing behavior began (Figures 5D–5F). Ca2+ signals in dmPFC neurons projecting to the PAG showed an increasing trend but did not reach statistical significance (Figures 5H–5J). In contrast, dmPFC neurons projecting to the aBLA showed a reduction in activity after pushing behavior began (Figures 5L–5N).

These Ca2+ dynamics suggest that during social competition, pushing behavior is associated with activation of dmPFC-DRN projection neurons related to victory, while dmPFC-aBLA projection neurons related to defeat are suppressed.

Figure 5: Activity Dynamics of Different dmPFC Projection Neuron Subgroups in the Tube Test

dmPFC aBLA-Projecting Layer 2/3 Neurons Inhibit Layer 5 Neurons

To understand how dmPFC neurons associated with victory and defeat coordinate their activity to drive behavior, the authors explored how they interact within the dmPFC (Figure 6).

When optogenetically activating victory-associated Layer 5 neurons projecting to the DRN and recording the activity of Layer 2/3 neurons projecting to the aBLA (Figure 6A), no significant effects were observed on the activity of these Layer 2/3 neurons after activation or inhibition (Figures 6B–6H).

When optogenetically activating defeat-associated Layer 2/3 neurons projecting to the aBLA and recording Layer 5 neurons (Figure 6I), it was found that 75% of the dmPFC Layer 5 pPYR neurons were inhibited, and 6% were activated (Figures 6J–6L). In contrast, when the aBLA-projecting neurons were optogenetically inhibited, 62% of the dmPFC Layer 5 pPYR neurons were activated, and only 6% were inhibited (Figures 6N–6P).

These results suggest that the dmPFC Layer 2/3 neurons projecting to the aBLA, referred to as "defeat neurons," significantly inhibit the "victory neurons" in Layer 5.

Figure 6: aBLA-Projecting Layer 2/3 Neurons in the dmPFC Inhibit Layer 5 Neurons

Defeat-Associated Neurons Inhibit Victory-Associated Neurons via GABAergic Interneurons

To understand the cellular mechanisms through which defeat-associated neurons inhibit victory-associated neurons, the authors conducted a brain slice patch-clamp experiment (Figure 7).

AAV expressing Cre-inducible light-sensitive ChR2 was injected into the dmPFC, and a retrograde virus (AAV2/Retro-hSyn-Cre) was injected into the aBLA to express ChR2 with an EGFP tag in neurons projecting to the aBLA. To visualize neurons projecting to the DRN, a retrograde virus (AAV2/Retro-hSyn-tdTomato-WPRE-pA) was injected into the DRN. A blue light pulse (5 ms pulse width) was used to stimulate neurons projecting to the aBLA in acute brain slices, while whole-cell patch-clamp recordings were made from neurons projecting to the DRN (Figure 7A).

The results showed that in the majority (73%) of neurons projecting to the DRN, the amplitude of eIPSCs was greater than that of eEPSCs (Figure 7B). Light-evoked EPSCs were completely blocked by the voltage-gated sodium channel blocker tetrodotoxin (TTX) and were restored with the application of TTX+4AP (the potassium channel blocker 4-aminopyridine) (Figure 7C), indicating that eEPSCs were mediated by direct synaptic connections. On the other hand, light-evoked IPSCs were also completely blocked by TTX, but were not restored by the application of TTX+4AP (Figure 7D), suggesting that eIPSCs were mediated by polysynaptic inhibitory currents. Furthermore, the GABAA receptor antagonist picrotoxin (PTX) blocked eIPSCs (Figure 7E).
Using optogenetic experiments to regulate the respective circuits in SST-Cre or PV-Cre mice, the results showed that both SST and PV interneurons were involved in the feedforward inhibition from Layer 2/3 to Layer 5 neurons in the dmPFC.

Figure 7: Defeat-Associated Neurons Inhibit Victory-Associated Neurons via GABAergic Interneurons

Summary

Through detailed neuroscience research, this article reveals the roles of different neural pathways in the mouse dorsomedial prefrontal cortex (dmPFC) during social competition, particularly the roles of the dmPFC-DRN and dmPFC-PAG pathways in promoting victory-associated behaviors, and the role of the dmPFC-aBLA pathway in promoting defeat-associated behaviors. This not only enhances our understanding of how the brain regulates social behaviors but also provides potential targets for the development of new treatments for social disorders and psychiatric diseases. It contributes to a deeper exploration of the complexity of brain function and the development of more effective intervention strategies.

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Tel： +86 18971216876

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Neuron︱Zhejiang University’s Hailan Hu Team Analyzes the Neural Circuits Behind Social Hierarchy in Mice

Downstream Brain Regions of the dmPFC Associated with Victory or Defeat

Inhibiting Different dmPFC Downstream Circuits Alters Dominance Rank

Activating Different dmPFC Downstream Circuits Alters Dominance Rank

Regulating the aBLA Affects Rank in the Tube Test

Activity Dynamics of Different dmPFC Projection Neuron Subgroups in the Tube Test

dmPFC aBLA-Projecting Layer 2/3 Neurons Inhibit Layer 5 Neurons

Defeat-Associated Neurons Inhibit Victory-Associated Neurons via GABAergic Interneurons

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Neuron︱Zhejiang University’s Hailan Hu Team Analyzes the Neural Circuits Behind Social Hierarchy in Mice

Downstream Brain Regions of the dmPFC Associated with Victory or Defeat

Inhibiting Different dmPFC Downstream Circuits Alters Dominance Rank

Activating Different dmPFC Downstream Circuits Alters Dominance Rank

Regulating the aBLA Affects Rank in the Tube Test

Activity Dynamics of Different dmPFC Projection Neuron Subgroups in the Tube Test

dmPFC aBLA-Projecting Layer 2/3 Neurons Inhibit Layer 5 Neurons

Defeat-Associated Neurons Inhibit Victory-Associated Neurons via GABAergic Interneurons

Summary

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