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.

Unilateral anterior crossbite (UAC) has been demonstrated to cause masseter hyperactivity via the periodontal trigeminal mesencephalic nucleus (Vme)–trigeminal motor nucleus circuit. Here, we studied activation of motor neurons of the facial nucleus (VII), hypoglossal nucleus (XII), nucleus ambiguus (Amb), and spinal nucleus of the accessory nerve (SNA) in rats with UAC via their similar connections with Vme. An anterograde tracer, biotinylated dextran amine (BDA), was injected into the Vme to identify the central axon terminals around the motor neurons of VII, XII, Amb, and SNA. The expression of vesicular glutamate transporter 1 (VGLUT1) in neurons of VII, XII, Amb, and SNA, and the expression of acetylcholinesterase (AChE) were measured in the stapedius, lingualis, palatopharyngeal, and sternocleidomastoid muscles. In BDA‐treated rats, many BDA‐labeled cell bodies in the Vme and terminals in VII, XII, Amb, and SNA were identified. Compared with control rats, rats with UAC showed higher expression of VGLUT1 in these nuclei, and statistically significantly higher expression of AChE in the stapedius, lingualis, and sternocleidomastoid muscles, but not in the palatopharyngeal muscle. These findings suggest that UAC activates orofacial, head, and cervical multimotor behaviors via connections between the Vme and the corresponding motor nuclei.