The Involvement of Spinal Trigeminal Nucleus-Hippocampal Dopaminergic Pathway in Malocclusion-Induced TMD-Related Depression via Putative DRD2/NF-κB/LEPR Signaling Axis

Technical Summary: The Involvement of Spinal Trigeminal Nucleus-Hippocampal Dopaminergic Pathway in Malocclusion-Induced TMD-Related Depression via Putative DRD2/NF-κB/LEPR Signaling Axis

Problem Statement
Temporomandibular disorders (TMD) are prevalent orofacial conditions frequently comorbid with depression. While clinical evidence links TMD pain and dysfunction to emotional distress, the central neural mechanisms connecting malocclusion-induced TMD to depressive phenotypes remain largely unelucidated. Specifically, the existence of direct neural projections between the spinal trigeminal nucleus (SpV), a key relay for orofacial nociception, and the hippocampus (HPC), a region critical for emotional regulation, and their specific contribution to TMD-related depression, has not been fully established. Furthermore, the downstream molecular signaling cascades involving dopaminergic mechanisms and leptin receptor (LEPR) pathways within this circuit are unknown.

Methodology
The study employed a multidisciplinary approach combining behavioral analysis, viral tracing, chemogenetics, tissue clearing, and molecular biology:

• Animal Models: A unilateral anterior crossbite (UAC) mouse model was established to induce TMD and associated depressive behaviors. C57BL/6 wild-type, DAT-Cre, and Ai140 transgenic mice were utilized.
• Behavioral Assessment: Depression-like behaviors were quantified using the Forced Swimming Test (FST), Sucrose Preference Test (SPT) for anhedonia, and Open Field Test (OFT) for anxiety-like exploration.
• Neural Circuit Mapping:
  • Viral Tracing: Adeno-associated viruses (AAV2/1) carrying Cre-dependent EGFP or chemogenetic constructs (hM4Di for inhibition, hM3Dq for activation) were stereotaxically injected into the SpV of DAT-Cre mice to label and manipulate dopaminergic neurons projecting to the HPC.
  • Tissue Clearing: Whole-brain clearing techniques were applied to Ai140 mice injected with Cre-dependent AAVs to visualize 3D neural projections spanning the SpV and HPC.
• Chemogenetic Manipulation: The functional role of the SpV-HPC circuit was tested by activating or inhibiting SpV dopaminergic neurons via intraperitoneal Clozapine N-oxide (CNO) administration.
• Molecular Analysis:
  • In Vivo: RT-qPCR and Western Blot (WB) were performed on hippocampal tissues to quantify mRNA and protein levels of Dopamine Receptor D2 (DRD2), Leptin Receptor (LEPR), and Nuclear Factor kappa-B (NF-κB) phosphorylation.
  • In Vitro: Mouse hippocampal HT22 neuronal cells were used to establish causal relationships. DRD2 was knocked down via siRNA or overexpressed via lentivirus. The role of the NF-κB pathway was investigated using Verbascoside (VB), a dual PKC/NF-κB inhibitor.
  • Therapeutic Intervention: AAV-mediated overexpression of DRD2 or LEPR was delivered directly into the hippocampus of UAC mice to assess behavioral rescue.

Key Results

1. Phenotypic Validation: UAC mice successfully developed TMD pathology (confirmed by micro-CT and histology showing osteoarthritis and increased osteoclast activity) and exhibited significant depressive-like behaviors, including increased immobility in the FST, reduced sucrose preference, and decreased center exploration in the OFT.
2. Identification of a Novel Circuit: Multimodal imaging (viral tracing, immunofluorescence, and tissue clearing) confirmed the existence of a direct dopaminergic projection from the SpV to the HPC. TH+ (tyrosine hydroxylase) and DAT+ (dopamine transporter) neurons in the SpV project to the HPC.
3. Functional Role of the Circuit:
   • Chemogenetic inhibition (hM4Di) of SpV dopaminergic neurons induced depressive-like behaviors in control mice.
   • Chemogenetic activation (hM3Dq) of this pathway reversed depressive-like behaviors in UAC mice, indicating that functional suppression of this circuit contributes to TMD-related depression.
4. Molecular Mechanism (DRD2/NF-κB/LEPR Axis):
   • UAC mice exhibited significant downregulation of DRD2 and LEPR in the hippocampus.
   • In vitro experiments demonstrated that DRD2 overexpression upregulated LEPR, while DRD2 knockdown downregulated LEPR.
   • This regulation was mediated by the phosphorylation of NF-κB. Inhibition of NF-κB phosphorylation (using VB) blocked the DRD2-induced upregulation of LEPR.
   • In vivo, AAV-mediated overexpression of either DRD2 or LEPR in the hippocampus of UAC mice significantly alleviated depressive-like behaviors, confirming the therapeutic potential of this axis.

Key Contributions

• Anatomical Discovery: The study provides the first anatomical evidence of a direct dopaminergic projection from the Spinal Trigeminal Nucleus to the Hippocampus.
• Causal Link: It establishes a causal relationship between the functional suppression of this SpV-HPC dopaminergic pathway and the development of depression-like behaviors in a malocclusion-induced TMD model.
• Molecular Pathway Elucidation: The research defines a specific downstream signaling axis (DRD2/NF-κB/LEPR) through which dopaminergic signaling regulates hippocampal function in the context of TMD-related depression.
• Therapeutic Validation: It demonstrates that restoring DRD2 or LEPR expression in the hippocampus is sufficient to reverse behavioral deficits, identifying these molecules as potential therapeutic targets.

Significance
The paper claims that these findings elucidate novel mechanistic insights into the neuropsychiatric comorbidities of TMD. By identifying a specific neural circuit (SpV-HPC) and a molecular signaling axis (DRD2/NF-κB/LEPR), the study offers a new framework for understanding how orofacial pain and malocclusion translate into central emotional dysregulation. The authors posit that this pathway represents a potential therapeutic target for intervening in TMD-related depression, moving beyond symptomatic pain management to address the underlying central neural mechanisms.