Potentiation of the lateral habenula-ventral tegmental area pathway underlines the susceptibility to depression in mice with chronic pain.

Chronic pain often develops severe mood changes such as depression. However, how chronic pain leads to depression remains elusive and the mechanisms determining individuals' responses to depression are largely unexplored. Here we found that depression-like behaviors could only be observed in 67.9% of mice with chronic neuropathic pain, leaving 32.1% of mice with depression resilience. We determined that the spike discharges of the ventral tegmental area (VTA)-projecting lateral habenula (LHb) glutamatergic (Glu) neurons were sequentially increased in sham, resilient and susceptible mice, which consequently inhibited VTA dopaminergic (DA) neurons through a LHbGlu-VTAGABA-VTADA circuit. Furthermore, the LHbGlu-VTADA excitatory inputs were dampened via GABAB receptors in a pre-synaptic manner. Regulation of LHb-VTA pathway largely affected the development of depressive symptoms caused by chronic pain. Our study thus identifies a pivotal role of the LHb-VTA pathway in coupling chronic pain with depression and highlights the activity-dependent contribution of LHbGlu-to-VTADA inhibition in depressive behavioral regulation.

Coexpression of VGLUT1 and VGLUT2 in precerebellar neurons in the lateral reticular nucleus of the rat.

Vesicular glutamate transporter (VGLUT) 1 and VGLUT2 have been reported to distribute complementally in most brain regions and have been assumed to define distinct functional elements. Previous studies have shown the expression of VGLUT1 mRNA and VGLUT2 mRNA in the lateral reticular nucleus (LRN), a key precerebellar nucleus sending mossy fibers to the cerebellum. In the present study, we firstly examined the coexpression of VGLUT1 and VGLUT2 mRNA in the LRN of the rat by dual-fluorescence in situ hybridization. About 81.89 % of glutamatergic LRN neurons coexpressed VGLUT1 and VGLUT2 mRNA, and the others expressed either VGLUT1 or VGLUT2 mRNA. We then injected the retrograde tracer Fluogold (FG) into the vermal cortex of cerebellum, and observed that 95.01 % and 86.80 % of FG-labeled LRN neurons expressed VGLUT1 or VGLUT2 mRNA respectively. We further injected the anterograde tracer biotinylated dextran amine (BDA) into the LRN, and found about 82.6 % of BDA labeled axon terminals in the granular layer of cerebellar cortex showed both VGLUT1- and VGLUT2-immunoreactivities. Afterwards, we observed under electron microscopy that anterogradely labeled axon terminals showing immunoreactivity for VGLUT1 or VGLUT2 made asymmetric synapses with dendritic profiles of cerebellar neurons. Finally, we selectively down-regulated the expression of VGLUT1 mRNA or VGLUT2 mRNA by using viral vector mediated siRNA transfection and detected that the fine movements of the forelimb of rats were disturbed. These results indicated that LRN neurons coexpressing VGLUT1 and VGLUT2 project to the cerebellar cortex and these neurons might be critical in mediating the forelimb movements.

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.

Collateral Projections from the Lateral Parabrachial Nucleus to the Central Amygdaloid Nucleus and the Ventral Tegmental Area in the Rat.

Lateral parabrachial nucleus (LPB) is a critical region in the integration and transmission of peripheral nociceptive information. The parabrachio-amygdaloid (P-Amy) pathway and parabrachio-ventral tegmental area (P-VTA) pathway is thought to be significant in regulation of pain-related negative emotions. In present study, retrograde tract tracers Fluoro-gold (FG) and tetramethylrhodramine-dextran (TMR) were stereotaxically injected into the right central amygdaloid nucleus (CeA) and right VTA, respectively. Then, part of these rats were performed with the spare nerve injury (SNI) in the controlateral side of FG and TMR injection. Afterwards, double- or triple-immunofluorescent histochemistry was used to examine FG/TMR double- and FG/TMR/FOS or FG/TMR/CGRP triple-labeled neurons in the LPB. The results showed that all of FG, TMR single- and FG/TMR double-labeled neurons were distributed in the LPB bilaterally with an ipsilateral predominance. The proportion of FG/TMR double-labeled neurons to the total number of FG- and TMR-labeled neurons was 10.78% and 13.07%, respectively. Nearly all of the FG/TMR double-labeled neurons (92.67%) showed calcitonin gene-related peptide (CGRP) immunopositive. On the other hand, in the SNI rats, about 89.49% and 77.87% of FG- and TMR-labeled neurons were FG/FOS- and TMR/FOS-positive neurons; about 93.33% of the FG/TMR double-labeled neurons were FOS-LI. Our results suggest that the part of CGRP immunopositive neurons in the LPB send projection fibers to both the CeA and VTA by the way of axon collaterals, which are activated by the nociceptive stimulation in the SNI condition, and may play an important role in the transmission of peripheral nociceptive information. Anat Rec, 302:1178-1186, 2019. © 2018 The Authors. The Anatomical Record published by Wiley Periodicals, Inc. on behalf of American Association of Anatomists.

VGLUT1 or VGLUT2 mRNA-positive neurons in spinal trigeminal nucleus provide collateral projections to both the thalamus and the parabrachial nucleus in rats.

The trigemino-thalamic (T-T) and trigemino-parabrachial (T-P) pathways are strongly implicated in the sensory-discriminative and affective/emotional aspects of orofacial pain, respectively. These T-T and T-P projection fibers originate from the spinal trigeminal nucleus (Vsp). We previously determined that many vesicular glutamate transporter (VGLUT1 and/or VGLUT2) mRNA-positive neurons were distributed in the Vsp of the adult rat, and most of these neurons sent their axons to the thalamus or cerebellum. However, whether VGLUT1 or VGLUT2 mRNA-positive projection neurons exist that send their axons to both the thalamus and the parabrachial nucleus (PBN) has not been reported. Thus, in the present study, dual retrograde tract tracing was used in combination with fluorescence in situ hybridization (FISH) for VGLUT1 or VGLUT2 mRNA to identify the existence of VGLUT1 or VGLUT2 mRNA neurons that send collateral projections to both the thalamus and the PBN. Neurons in the Vsp that send collateral projections to both the thalamus and the PBN were mainly VGLUT2 mRNA-positive, with a proportion of 90.3%, 93.0% and 85.4% in the oral (Vo), interpolar (Vi) and caudal (Vc) subnucleus of the Vsp, respectively. Moreover, approximately 34.0% of the collateral projection neurons in the Vc showed Fos immunopositivity after injection of formalin into the lip, and parts of calcitonin gene-related peptide (CGRP)-immunopositive axonal varicosities were in direct contact with the Vc collateral projection neurons. These results indicate that most collateral projection neurons in the Vsp, particularly in the Vc, which express mainly VGLUT2, may relay orofacial nociceptive information directly to the thalamus and PBN via axon collaterals.

The synergistic effect of treatment with triptolide and MK-801 in the rat neuropathic pain model.

Triptolide (T10), an active component of Tripterygium wilfordii Hook F, is reported to have potent anti-inflammatory and analgesic effects. Additionally, MK-801, a noncompetitive N-methyl-D-aspartate receptor antagonist, can reduce glutamate toxicity and has a significant analgesic effect on chronic pain. In this study, we tested the possible synergistic analgesic ability by intrathecal administration of T10 and MK-801 for the treatment of neuropathic pain. Single T10 (3, 10, or 30 µg/kg), MK-801 (10, 30, or 90 µg/kg), or a combination of them were intrathecally administrated in rats with spinal nerve ligation. We found that single administration of T10 caused a slow-acting but long-term analgesic effect, while single administration of MK-801 caused a fast-acting but short-term effect. Administration of their combination showed obviously synergic analgesia and the 1:3 ratio of T10 to MK-801 reached the peak effect. Furthermore, application of T10 and/or MK-801 significantly inhibited the activation of microglia and astrocyte and phosphorylation of STAT3 and NR2B in the spinal dorsal horn induced by chronic neuropathic pain. Our data suggest that the combination of T10 and MK-801 may be a potentially novel strategy for treatment of neuropathic pain.

Melatonin Suppresses Neuropathic Pain via MT2-Dependent and -Independent Pathways in Dorsal Root Ganglia Neurons of Mice.

Melatonin (Mel) and its receptors (MT1 and MT2) have a well-documented efficacy in treating different pain conditions. However, the anti-nociceptive effects of Mel and Mel receptors in neuropathic pain (NP) are poorly understood. To elucidate this process, pain behaviors were measured in a dorsal root ganglia (DRG)-friendly sciatic nerve cuffing model. We detected up-regulation of MT2 expression in the DRGs of cuff-implanted mice and its activation by the agonist 8-M-PDOT (8MP). Also, Mel attenuated the mechanical and thermal allodynia induced by cuff implantation. Immunohistochemical analysis demonstrated the expression of MT2 in the DRG neurons, while MT1 was expressed in the satellite cells. In cultured primary neurons, microarray analysis and gene knockdown experiments demonstrated that MT2 activation by 8MP or Mel suppressed calcium signaling pathways via MAPK1, which were blocked by RAR-related orphan receptor alpha (RORα) activation with a high dose of Mel. Furthermore, expression of nitric oxide synthase 1 (NOS1) was down-regulated upon Mel treatment regardless of MT2 or RORα. Application of Mel or 8MP in cuff-implanted models inhibited the activation of peptidergic neurons and neuro-inflammation in the DRGs by down-regulating c-fos, calcitonin gene-related peptide [CGRP], and tumor necrosis factor-1α [TNF-1α] and interleukin-1ß [IL-1ß]. Addition of the MT2 antagonist luzindole blocked the effects of 8MP but not those of Mel. In conclusion, only MT2 was expressed in the DRG neurons and up-regulated upon cuff implantation. The analgesic effects of Mel in cuff-implanted mice were closely associated with both MT2-dependent (MAPK-calcium channels) and MT2-independent (NOS1) pathways in the DRG.

Descending control of itch transmission by the serotonergic system via 5-HT1A-facilitated GRP-GRPR signaling.

Central serotonin (5-hydroxytryptophan, 5-HT) modulates somatosensory transduction, but how it achieves sensory modality-specific modulation remains unclear. Here we report that enhancing serotonergic tone via administration of 5-HT potentiates itch sensation, whereas mice lacking 5-HT or serotonergic neurons in the brainstem exhibit markedly reduced scratching behavior. Through pharmacological and behavioral screening, we identified 5-HT1A as a key receptor in facilitating gastrin-releasing peptide (GRP)-dependent scratching behavior. Coactivation of 5-HT1A and GRP receptors (GRPR) greatly potentiates subthreshold, GRP-induced Ca(2+) transients, and action potential firing of GRPR(+) neurons. Immunostaining, biochemical, and biophysical studies suggest that 5-HT1A and GRPR may function as receptor heteromeric complexes. Furthermore, 5-HT1A blockade significantly attenuates, whereas its activation contributes to, long-lasting itch transmission. Thus, our studies demonstrate that the descending 5-HT system facilitates GRP-GRPR signaling via 5-HT1A to augment itch-specific outputs, and a disruption of crosstalk between 5-HT1A and GRPR may be a useful antipruritic strategy.