Effect of dental malocclusion on cerebellar neuron activation via the dorsomedial part of the principal sensory trigeminal nucleus.

Occlusion has been proposed to play a role for body posture and balance, both of which are mediated mainly by the cerebellum. The dorsomedial part of the principal sensory trigeminal nucleus (Vpdm) has direct projection to the cerebellum. The experimental unilateral anterior crossbite (UAC) has an impact on the motor nuclei in the brain stem via trigeminal mesencephalic nucleus (Vme). The current aim was to explore whether UAC has an impact on Vpdm-cerebellum circuit. The inferior alveolar nerve was injected into cholera toxin B subunit (CTb), the cerebellum was injected into fluoro-gold (FG), and the Vpdm was injected into biotinylated dextran amine (BDA) to identify the activation of Vpdm-cerebellum circuit by UAC. Data indicated that there were more neuronal nuclei (NeuN)/CTb/FG triple-labelled neurons and NeuN/CTb/vesicular glutamate transporter 1(VGLUT1) triple-labelled neurons in the Vpdm, and more NeuN/BDA/ VGLUT1 triple-labelled neurons in the cerebellum of rats with UAC than in control rats. The VGLUT1 expression in the Vpdm and cerebellum in the UAC group was higher than that in control rats. These findings indicate an excitatory impact of UAC on the Vpdm-cerebellum pathway and support the role of occlusion for body posture and balance.

Proprioceptive mechanisms in occlusion-stimulated masseter hypercontraction.

Neurons in the trigeminal mesencephalic nucleus (Vme) have an axon that branches peripherally to innervate the orofacial region and projects centrally to the trigeminal motor nucleus (Vmo). They function as the primary neurons conveying proprioceptive messages. The present study aimed to demonstrate the presence of a periodontal-Vme-Vmo circuit and to provide evidence for its involvement in an experimental unilateral anterior crossbite (UAC) model, which can induce osteoarthritis in the temporomandibular joint. Cholera toxin B subunit (CTb) was injected into the inferior alveolar nerve of rats to help identify the central axon terminals of Vme neurons in the Vmo. The levels of vesicular glutamate transporter 1 (VGLUT1) expressed in the periodontal region, Vme, Vmo, and masseter, and the level of acetylcholinesterase (AChE) expressed in the masseter, were assessed in UAC rats and controls. In CTb-treated rats, many CTb-labeled cell bodies and endings were identified in the Vme and in the Vmo, respectively. In UAC rats, VGLUT1 was expressed at a statistically significantly higher level in the periodontal ligament, Vme, Vmo, and masseter than it was in control rats. The level of AChE protein was 1.97 times higher in UAC rat masseter compared with control rat masseter. These findings reveal a trigeminal mechanism underlying masseter hyperactivity induced by an altered occlusion.

Activation of the VpdmVGLUT1-VPM pathway contributes to anxiety-like behaviors induced by malocclusion.

Occlusal disharmony has a negative impact on emotion. The mesencephalic trigeminal nucleus (Vme) neurons are the primary afferent nuclei that convey proprioceptive information from proprioceptors and low-threshold mechanoreceptors in the periodontal ligament and jaw muscles in the cranio-oro-facial regions. The dorsomedial part of the principal sensory trigeminal nucleus (Vpdm) and the ventral posteromedial nucleus (VPM) of thalamus have been proven to be crucial relay stations in ascending pathway of proprioception. The VPM sends numerous projections to primary somatosensory areas (SI), which modulate emotion processing. The present study aimed to demonstrate the ascending trigeminal-thalamic-cortex pathway which would mediate malocclusion-induced negative emotion. Unilateral anterior crossbite (UAC) model created by disturbing the dental occlusion was applied. Tract-tracing techniques were used to identify the existence of Vme-Vpdm-VPM pathway and Vpdm-VPM-SI pathway. Chemogenetic and optogenetic methods were taken to modulate the activation of VpdmVGLUT1 neurons and the Vpdm-VPM pathway. Morphological evidence indicated the involvement of the Vme-Vpdm-VPM pathway, Vpdm-VPM-SI pathway and VpdmVGLUT1-VPM pathway in orofacial proprioception in wild-type mice and vesicular glutamate transporter 1 (VGLUT1): tdTomato mice, respectively. Furthermore, chemogenetic inhibition of VpdmVGLUT1 neurons and the Vpdm-VPM pathway alleviated anxiety-like behaviors in a unilateral anterior crossbite (UAC) model, whereas chemogenetic activation induced anxiety-like behaviors in controls and did not aggravate these behaviors in UAC mice. Finally, optogenetic inhibition of the VpdmVGLUT1-VPM pathway in VGLUT1-IRES-Cre mice reversed UAC-induced anxiety comorbidity. In conclusion, these results suggest that the VpdmVGLUT1-VPM neural pathway participates in the modulation of malocclusion-induced anxiety comorbidity. These findings provide new insights into the links between occlusion and emotion and deepen our understanding of the impact of occlusal disharmony on brain dysfunction.

Excitatory Impact of Dental Occlusion on Dorsal Motor Nucleus of Vagus.

Neurons in the trigeminal mesencephalic nucleus (Vme) have axons that branch peripherally to innervate the orofacial region and project centrally to several motor nuclei in brainstem. The dorsal motor nucleus of vagus nerve (DMV) resides in the brainstem and takes a role in visceral motor function such as pancreatic exocrine secretion. The present study aimed to demonstrate the presence of Vme-DMV circuit, activation of which would elicit a trigeminal neuroendocrine response. A masticatory dysfunctional animal model termed unilateral anterior crossbite (UAC) model created by disturbing the dental occlusion was used. Cholera toxin B subunit (CTb) was injected into the inferior alveolar nerve of rats to help identify the central axon terminals of Vme neurons around the choline acetyltransferase (ChAT) positive motor neurons in the DMV. The level of vesicular glutamate transporter 1 (VGLUT1) expressed in DMV, the level of acetylcholinesterase (AChE) expressed in pancreas, the level of glucagon and insulin expression in islets and serum, and the blood glucose level were detected and compared between UAC and the age matched sham-operation control mice. Data indicated that compared with the controls, there were more CTb/VGLUT1 double labeled axon endings around the ChAT positive neurons in the DMV of UAC groups. Mice in UAC group expressed a higher VGLUT1 protein level in DMV, AChE protein level in pancreas, glucagon and insulin level in islet and serum, and higher postprandial blood glucose level, but lower fasting blood glucose level. All these were reversed at 15-weeks when UAC cessation was performed from 11-weeks (all, P < 0.05). Our findings demonstrated Vme-DMV circuit via which the aberrant occlusion elicited a trigeminal neuroendocrine response such as alteration in the postprandial blood glucose level. Dental occlusion is proposed as a potential therapeutic target for reversing the increased postprandial glucose level.

Malocclusion Generates Anxiety-Like Behavior Through a Putative Lateral Habenula-Mesencephalic Trigeminal Nucleus Pathway.

Malocclusion is an important risk factor for temporomandibular disorder (TMD), a series of disorders characterized by dysfunction in the orofacial region involving the temporomandibular joint (TMJ) and jaw muscles. We recently showed that experimental unilateral anterior crossbite (UAC) produced masseter hyperactivity through a circuit involving the periodontal proprioception, trigeminal mesencephalic nucleus (Vme), and trigeminal motor nucleus (Vmo). Anxiety is a common complication in patients with TMD. The lateral habenula (LHb) is involved in emotional modulation and has direct projections to the Vme. Therefore, the present research examined whether UAC facilitates excitatory input from the LHb to the Vme and, subsequently, anxiety-like behaviors in rats. The LHb activation was evaluated by the electrophysiological recording, assessment of vesicular glutamate transporter-2 (VGLUT2) mRNA expression, and measurement of anxiety-like behaviors. The effects of LHb activity on Vme were evaluated by electrophysiological recording from Vme neurons and local changes in VGLUT2 protein density. UAC produced anxiety in modeled rats and increased neuronal activity in the LHb. VGLUT2 mRNA expression was also increased in the LHb. Further, VGLUT2-positive boutons were observed in close apposite upon parvalbumin (PV)-labeled Vme neurons. VGLUT2 protein expression was also increased in the Vme. Significantly, injection of VGLUT2-targeted shRNA into the LHb reduced the expression of VGLUT2 protein in the Vme, attenuated UAC-associated anxiety-like behaviors, and attenuated electrophysiological changes in the Vme neurons. In conclusion, we show that UAC activates the LHb neurons as well as the periodontal proprioceptive pathway to provide excitatory input to the Vme and produce anxiety in rats. These findings provide a rationale for suppressing activity of the LHb to attenuate both the physical and psychological effects of TMD.

Efferent and afferent connections of GABAergic neurons in the supratrigeminal and the intertrigeminal regions. An immunohistochemical tract-tracing study in the GAD67-GFP knock-in mouse.

It has been reported in the cat and rat that inhibitory premotor neurons, which send their axons to motoneurons of the trigeminal motor nucleus (Vm) are distributed in the reticular regions around the Vm, especially in the supratrigeminal region (Vsup) and the intertrigeminal region (Vint). In the present study, we examined neuronal connections of GABAergic neurons in the Vsup and Vint in the mouse by utilizing the adult heterozygous GAD67-GFP knock-in mouse, in which green fluorescence protein (GFP) is expressed in GABAergic neurons under the control of the endogenous GAD (GAD67) gene promoter [Yanagawa, Y., Kaneko, K., Kanbara, N., Totsuka, M., Yagi, T., Obata, K., 2001. Development of mouse expressing GFP in GABAergic neurons. Neurosci. Res. Suppl. 25, S77; Tamamaki, N., Yanagawa, Y., Tomioka, R., Miyazaki, J.-I., Obata, K., Kaneko, T., 2003. Green fluorescent protein expression and colocalization with calretinin, parvalbumin and somatostatin in the GAD67-GFP knock-in mouse. J. Comp. Neurol. 467, 60-79]. The connections were examined light- and electron-microscopically by combining the anterograde or the retrograde tract-tracing method with the immunohistochemical method for GFP. The data indicated that the Vsup and Vint of the mouse contained GABAergic neurons, which received projection fibers from the marginal layer of the nucleus of the spinal tract of the trigeminal nerve (Vc) on the ipsilateral side and sent their axons to the Vm on the contralateral side. Some of these GABAergic neurons may represent Vm-premotor neurons that receive nociceptive input from the Vc to elicit jaw-opening reflex by inhibiting jaw-closing Vm-motoneurons.

Distribution of gephyrin-immunoreactivity in the trigeminal motor nucleus: an immunohistochemical study in rats.

It has been established that a postsynaptic scaffolding protein, gephyrin, is essential for anchoring two main groups of inhibitory receptors, GABA(A) receptors (GABA(A) Rs) and glycine receptors (GlyRs), to the postsynaptic sites of neurons. The present study was primarily attempted to examine if expression patterns of gephyrin might be different between jaw-closing (JC) and jaw-opening (JO) motoneurons. The JC- and JO-motoneurons in the rat trigeminal motor nucleus (Vm) were located in the dorsolateral (Vm.dl) and ventromedial (Vm.vm) divisions, respectively (Mizuno et al.,1975). Thus, immunoreactivity (IR) for gephyrin was investigated in the Vm: immunofluorescence histochemistry for gephyrin was combined with retrograde tract-tracing of fluorogold (FG), which was injected into nerves innervating JC-muscles or nerves innervating JO-muscles; neuronal cells were counterstained with propidium iodide (PI). The Vm.dl was discriminated from the Vm.vm by the presence of vesicular glutamate transporter 1 (VGLUT1)-immunopositive axon terminals, which were distributed in the Vm.dl but not in the Vm.vm (Pang et al., J Comp Neurol 2009;512:595-612). Gephyrin-IR showed a punctate pattern of fluorescence, and motoneuronal profiles were coated with small clusters of gephyrin-immunopositive puncta throughout the Vm. The distribution density of such clusters was apparently higher in the Vm.dl than in the Vm.vm; this was confirmed quantitatively by a method similar to that described by Lorenzo et al. (Eur J Neurosci 2006;23:3161-3170). On the basis of the present results, possible correlation between the distribution density of gephyrin clusters in the submembrane region of Vm motoneurons and that of axon terminals making inhibitory synapses on Vm motoneurons was discussed.

Axon terminals expressing vesicular glutamate transporter VGLUT1 or VGLUT2 within the trigeminal motor nucleus of the rat: origins and distribution patterns.

Little is known about the significance of the two types of glutamatergic neurons (those expressing vesicular glutamate transporter VGLUT1 or VGLUT2) in the control of jaw movements. We thus examined the origin and distribution of axon terminals with VGLUT1 or VGLUT2 immunoreactivity within the trigeminal motor nucleus (Vm) in the rat. The Vm was divided into the dorsolateral division (Vm.dl; jaw-closing motoneuron pool) and the ventromedial division (Vm.vm; jaw-opening motoneuron pool). VGLUT1-immunopositive terminals were seen within the Vm.dl only, whereas VGLUT2-immunopositive ones were distributed to both the Vm.dl and the Vm.vm. Transection of the motor root eliminated almost all VGLUT1-immunopositive axons in the Vm.dl, with no changes of VGLUT2 immunoreactivity in the two divisions, indicating that the VGLUT1- and VGLUT2-immunopositive axons came from primary afferents in the mesencephalic trigeminal nucleus and premotor neurons for the Vm, respectively. In situ hybridization histochemistry revealed that VGLUT2 neurons were much more numerous than VGLUT1 neurons in the regions corresponding to the reported premotoneuron pool for the Vm. The results of immunofluorescence labeling combined with anterograde tract tracing further indicated that premotor neurons with VGLUT2 in the trigeminal sensory nuclei, the supratrigeminal region, and the reticular region ventral to the Vm sent axon terminals contacting trigeminal motoneurons and that some of the VGLUT1-expressing premotor neurons in the reticular region ventral to the Vm sent axon terminals to jaw-closing motoneurons. The present results suggested that the roles played by glutamatergic neurons in controlling jaw movements might be different between VGLUT1- and VGLUT2-expressing neurons.