Mol. Psychiatry | Team Led by Miao He and Ling Gong Reveals Independent Circuits and Behavioral Regulation Mechanisms of Two Neuronal Subtypes in the Medial Mammillary Body

Differences in Spontaneous Firing Between _MMCB and _MMPV Neurons

In previous whole-cell patch-clamp recordings, the authors observed that some MM neurons exhibited two distinct patterns of spontaneous firing: tonic and bursting. To further investigate this phenomenon, they switched to cell-attached recordings and found that _MMCB neurons had a lower rate of spontaneous activity compared to _MMPV neurons, but a higher incidence of burst firing. Some MM neurons were capable of switching between tonic and bursting modes.

N — methyl-D-aspartate receptors (NMDARs) and T-type voltage-sensitive calcium channels (T-VSCCs) have been reported to play key roles in neuronal burst firing across various brain regions. The authors further examined this and found that activation of NMDARs promoted the transition of most tonic-firing neurons into a bursting mode. Notably, _MMCB neurons generated more action potentials per burst with longer inter-burst intervals. Additionally, _MMCB neurons exhibited a higher incidence of post-hyperpolarization rebound firing mediated by T-VSCCs, consistent with their bursting characteristics.

Figure 3. Differences in spontaneous firing between _MMCB and _MMPV neurons.

 

Segregated Outputs and Differential Inputs of _MMCB and _MMPV Neurons

To visualize the output projections of _MMCB and _MMPV  neurons, the researchers injected AAV-DIO-ChR2-RFP and AAV-fDIO-ChR2-GFP into the MM region of CBcre::PVFlp mice. The results showed that the axon terminals of _MMCB and _MMPV  neurons exhibited minimal overlap in downstream brain regions such as the anterior thalamus (AT), ventral tegmental nucleus (VTg), and retrotegmental pontine nucleus (RtTgP). This indicates that _MMCB and _MMPV neurons form anatomically segregated output circuits, each establishing topographically distinct subcircuits with their respective downstream targets.

Furthermore, the team used rabies virus-based tracing to label the inputs to MM neurons. In CBcre::PVFlp mice, helper viruses (AAV-DIO-GFP-TVA and AAV-DIO-G) were injected into the MM, and after two weeks, EnvA-RVΔG-RFP was injected into the AT region. The study found that compared to _MMPV  neurons, _MMCB neurons received relatively fewer inputs from the hippocampus and more from the hypothalamus. Both neuron types received their strongest input from the subiculum (SUB), but with distinct topographic preferences: _MMCB favored the ventral subiculum (vSUB), while _MMPV  favored the dorsal subiculum (dSUB), consistent with previously observed dorsoventral patterns of SUB-to-MM projections.

Figure 4. _MMCB  and _MMPV Project to Complementary Brain Regions and Receive Differential Inputs

 

_MMCB and _MMPV Mediate Distinct Behavioral Responses

To further determine whether _MMCB and _MMPV neurons elicit different behavioral effects, the researchers used optogenetic techniques by injecting AAV-DIO-ChR2-RFP into the MM region of CBcre::PVcre mice. Combining this with real-time place preference and open field tests, they found that only activation of _MMCB induced avoidance behavior and enhanced locomotion, while _MMPV activation had no such effect.

Additionally, by injecting a calcium imaging virus (AAV-DIO-GCaMP7) into the MM region, the researchers found that calcium activity in _MMCB neurons significantly increased around movement onset, with movement-related intensity 65% higher than that of _MMPV neurons, which showed no significant change. These results align with the optogenetic findings, further confirming that _MMCB—but not _MMPV—plays a key role in motor regulation, and highlighting the functional divergence of molecularly defined neuronal subtypes.

Figure 5. Optogenetic activation of _MMCB—but not _MMPV—induces place aversion and promotes locomotion.


Figure 6. _MMCB and _MMPV mediate distinct movement-related activity.

 

_MMCB and _MMPV Form Parallel Circuits Without Local Interactions

To directly assess whether _MMCB and _MMPV neurons are interconnected, the researchers performed whole-cell patch-clamp recordings to measure postsynaptic responses following optogenetic activation of either _MMCB or _MMPV neurons. Interestingly, while direct activation could be readily detected in ChR2-expressing neurons, no optogenetically evoked postsynaptic currents (oPSCs) were observed in ChR2-negative neurons. To rule out potential NMDAR inactivation by Mg²⁺, Mg²⁺ was removed from the ACSF, yet still no oPSCs were detected in ChR2-negative neurons.

These findings further confirm that there are no synaptic connections or cross-activation between _MMCB and _MMPV neurons, indicating that they form independent, parallel neuronal subcircuits. Among the recorded ChR2-negative cells, some were located in the same genetically defined region as neighboring ChR2-positive cells, yet still showed no evidence of direct activation—suggesting that the lack of local connectivity among neurons within the MM is a general feature.

Figure 7. No Cross-Activation Observed Between _MMCB and _MMPV Neurons

 

Conclusion

This study is the first to systematically identify two distinct neuronal subtypes in the medial mammillary body, marked by CB and PV, and to reveal their independent, parallel circuit mechanisms and roles in behavioral regulation. The lack of local connectivity in the MM suggests that its functions are primarily mediated through long-range circuits. This research provides a cell type–specific framework for understanding the functional organization of the MM, the mechanisms underlying neuropsychiatric disorders, and potential targets for therapeutic intervention.

The viral tools used in this study were provided by Brain Case Biotech.

 

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