PhD Candidate Sun Yat-sen Memorial Hospital, Sun Yat-sen University
Objectives Trigeminal nerve stimulation (TNS) is a promising noninvasive neuromodulation therapy for depression; however, its mechanisms remain unclear. This study investigated antidepressant-like effects of TNS and its underlying mechanisms in post-traumatic brain injury depression (PTD).
Design The antidepressant efficacy of TNS was evaluated in multiple depression models using standardized behavioral assays. Peripheral mechanisms were probed by chemogenetic activation or inhibition of trigeminal ganglion (TG) neuronal subpopulations in Cre-line mice. Centrally, pathway-selective chemogenetic manipulations tested the role of the paraventricular nucleus–ventral tegmental area–nucleus accumbens (PVN-VTA-NAc) circuit, coupled with in vivo (GPCR)-activation-based dopamine (GRAB-DA) fluorescence monitoring to quantify dopamine dynamics. Receptor-specific contributions were assessed using shRNA knockdown of D1/D2 or D3 receptors (D1/D2 or D3) in the NAc. Cerebrospinal fluid (CSF) cytokine profiling was used to evaluate inflammatory modulation. Primary endpoints were analyzed after the 10-day TNS treatment and following a 10-day post-stimulation period.
Results TNS alleviated depressive-like behaviors across models. TG TRPV1⁺ neurons are both necessary and sufficient for efficacy. Chemogenetic modulation of the PVN-VTA-NAc pathway reproduced or abolished TNS effects, while GRAB-DA recordings confirmed increased dopamine release during stimulation. In the NAc, D1/D2 knockdown impaired effects of treatment, whereas D3 knockdown eliminated persistence after stimulation. TNS reduced CSF cytokines in controls and D1/D2 knockdown mice, but not in D3 knockdown mice, indicating that early D3-dependent anti-inflammatory effects sustain long-term benefits.
Conclusions TNS alleviates depressive-like behaviors through TG TRPV1⁺ neurons and PVN-VTA-NAc circuit activation, with D1/D2 receptors mediating acute effects and D3 receptors maintaining lasting anti-inflammatory benefits.
Objectives
Post-traumatic brain injury depression (PTD) represents a severe and treatment-resistant subtype of depression, for which current antidepressant therapies exhibit limited efficacy, delayed onset, and substantial side effects. Trigeminal nerve stimulation (TNS) has recently emerged as a promising noninvasive neuromodulatory therapy, demonstrating antidepressant effects in both preclinical and clinical studies. However, the mechanisms by which peripheral trigeminal inputs access and modulate central mood-regulating circuits remain unclear. The trigeminal ganglion (TG) contains heterogeneous sensory neuron subtypes that may serve as an entry point for peripheral neuromodulation. The present study investigated whether specific TG neuronal populations mediate TNS effects and whether TNS can activate the paraventricular nucleus (PVN)–ventral tegmental area (VTA)–nucleus accumbens (NAc) circuit to enhance dopaminergic signaling and alleviate depressive-like behaviors. We further examined the contribution of dopamine receptor subtypes to acute and long-term antidepressant effects of TNS.
Methods
Male adult mice were subjected to three validated depression models: lipopolysaccharide (LPS)-induced inflammation, chronic restraint stress (CRS), and PTD induced by weight-drop traumatic brain injury. TNS was administered daily using clinically relevant parameters (40 Hz, 0.2 mA, 200 μs pulses; 30 s/min duty cycle) via electrodes targeting the infraorbital branch of the trigeminal nerve. Depressive-like behaviors were assessed using the open field test (OFT), and sucrose preference test (SPT), tail suspension test (TST).
To identify TG neuron subtypes mediating TNS effects, chemogenetic activation or inhibition (hM3Dq/hM4Di) was targeted to Piezo2-, Tac1-, or TRPV1-expressing TG neurons via stereotaxic AAV delivery. Neuronal activity was validated using whole-cell patch-clamp recordings in TG neuron cultures.
Central circuit mechanisms were examined using viral tracing, intersectional chemogenetic manipulation of the PVN–VTA–NAc pathway, optogenetic activation of PVN terminals in the VTA, and electrophysiology. Neuronal activation was assessed using ΔFosB immunostaining and in vivo calcium imaging (GCaMP6s in DAT-Cre mice). Dopamine dynamics in the NAc were monitored via GRAB-DA fiber photometry. Dopamine receptor involvement was tested using NAc-targeted shRNA knockdown of D1/D2 or D3 receptors, complemented by pharmacological receptor blockade. Cytokine levels in cerebrospinal fluid were analyzed using a cytokine array to evaluate neuroinflammatory responses. Statistical analyses were conducted using t-tests or ANOVA with significance set at p< 0.05.
Results
TNS alleviates depressive-like behaviors across multiple models
TNS produced robust antidepressant effects in LPS, CRS, and PTD mice. In all three models, TNS increased OFT center exploration, restored sucrose preference, and reduced TST immobility without altering total locomotor activity.
TG TRPV1⁺ neurons are required for TNS antidepressant actions
Chemogenetic activation of TRPV1⁺ TG neurons replicated the behavioral benefits of TNS, whereas activation of Piezo2- or Tac1-expressing neurons did not. Inhibition of TG TRPV1⁺ neurons abolished TNS efficacy across all depression models. These findings demonstrate that TRPV1⁺ TG neurons constitute the principal peripheral sensory population mediating TNS antidepressant effects.
TNS activates VTA dopaminergic neurons and restores dopamine levels
LC-MS analysis revealed widespread neurotransmitter alterations in PTD mice, including reduced dopamine levels that were restored by TNS. ΔFosB expression increased markedly in the VTA following TNS, predominantly in tyrosine hydroxylase-positive neurons, indicating specific activation of dopaminergic cells. In vivo calcium imaging further confirmed rapid, transient activation of VTA dopamine neurons in response to TNS, whereas control somatosensory stimulation did not elicit such activity.
The PVN–VTA–NAc pathway is necessary and sufficient for TNS efficacy
Chemogenetic activation of the PVN–VTA–NAc circuit in PTD mice replicated the antidepressant effects of TNS. Conversely, inhibiting VTA neurons within this circuit abolished TNS-induced behavioral improvements. Optogenetic stimulation of PVN terminals in the VTA elicited dopamine release in the NAc, demonstrating functional connectivity. TNS similarly induced robust GRAB-DA fluorescence increases in the NAc, which were blocked by haloperidol, confirming dopaminergic dependence.
Dopaminergic modulation by TNS may involve the PVN–VTA–NAc circuit
Optogenetic activation of PVN terminals in the VTA elicited transient increases in NAc GRAB-DA fluorescence, demonstrating functional PVN control over mesolimbic dopamine output. TNS similarly induced robust dopamine release in the NAc, which was absent in EGFP controls and eliminated by haloperidol, confirming dopaminergic specificity. These findings indicate that TNS enhances mesolimbic dopamine release through engagement of the PVN–VTA–NAc pathway.
D1/D2 and D3 dopamine receptors mediate distinct phases of TNS effects
Knockdown or pharmacological blockade of D1/D2 receptors diminished the acute antidepressant effects of TNS during the treatment period but did not affect post-treatment outcomes. In contrast, D3 receptor knockdown preserved acute improvement yet eliminated sustained antidepressant effects measured one week after cessation of TNS. Cytokine profiling revealed that TNS reduced inflammatory cytokines in control and D1/D2 knockdown mice but failed to do so in D3 knockdown mice, implicating D3 receptors in long-term modulation of neuroinflammation.
Conclusion
Our findings demonstrate that TNS alleviates depression-like behaviors by recruiting a peripheral–central neuromodulatory pathway involving TG TRPV1+ neurons and the PVN-VTA-NAc circuit. Moreover, we identified a temporal dissociation in dopamine receptor involvement: D1/D2 primarily mediates acute dopaminergic signaling during stimulation, whereas D3 receptors are required to sustain long-term therapeutic benefits, partly through neuroinflammation. Together, these results revealed a dual mechanism through which TNS exerts both rapid and enduring antidepressant effects, providing a mechanistic framework for its application in treatment-resistant depression.
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