The comorbidity of anxiety and depression frequently occurs in patients with neuropathic pain. The ventrolateral orbital cortex (VLO) plays a critical role in mediating neuropathic pain and anxiodepression in rodents. Previous studies suggested that 5-HT6 receptors in the VLO are involved in neuropathic pain. Strong evidence supports a close link between 5-HT6 receptors and affective disorders such as depression and anxiety disorders. However, it remains unclear whether the 5-HT6 receptors in the VLO are involved in neuropathic pain-induced anxiodepression. Using a rat neuropathic pain model of spared nerve injury (SNI), we demonstrated that rats exhibited significant anxiodepression-like behaviors and the expression of VLO 5-HT6 receptors obviously decreased four weeks after SNI surgery. Microinjection of the 5-HT6 receptor agonist EMD-386088 into the VLO or overexpression of VLO 5-HT6 receptors alleviated anxiodepression-like behaviors. These effects were blocked by pre-microinjection of a selective 5-HT6 receptor antagonist (SB-258585) or inhibitors of AC (SQ-22536), PKA (H89), and MEK1/2 (U0126) respectively. Meanwhile, the expression of p-ERK, p-CREB, and BDNF in the VLO decreased four weeks after SNI surgery. Furthermore, administration of EMD-386088 upregulated the expression of BDNF, p-ERK, and p-CREB in the VLO of SNI rats, which were reversed by pre-injection of SB-258585. These findings suggest that activating 5-HT6 receptors in the VLO has anti-anxiodepressive effects in rats with neuropathic pain via activating AC-cAMP-PKA-MERK-CREB-BDNF signaling pathway. Accordingly, 5-HT6 receptor in the VLO could be a potential target for the treatment of the comorbidity of neuropathic pain and anxiodepression.

The ventrolateral orbital cortex (VLO) is identified as an integral component of the endogenous analgesic system comprising a spinal cord - thalamic nucleus submedius - VLO - periaqueductal gray (PAG) - spinal cord loop. The present study investigates the effects of 5-HT5A receptor activation in the VLO on allodynia induced by spared nerve injury and formalin-evoked flinching behavior and spinal c-Fos expression in male SD rats, and further examines whether GABAergic modulation is involved in the effects evoked by VLO 5-HT5A receptor activation. We found an upregulation of 5-HT5A receptor expression in the VLO during neuropathic and inflammatory pain states. Microinjection of the non-selective 5-HT5A receptor agonist 5-CT into the VLO dose dependently alleviated allodynia, and flinching behavior and spinal c-Fos expression, which were blocked by the selective 5-HT5A receptor antagonist SB-699551. Moreover, application of the GABAA receptor antagonist bicuculline in the VLO augmented the analgesic effects induced by 5-CT in neuropathic and inflammatory pain states, whereas the GABAA receptor agonist muscimol attenuated these analgesic effects. Additionally, the 5-HT5A receptors were found to be colocalized with GABAergic neurons in the VLO. These results provide new evidence for the involvement of central 5-HT5A receptors in the VLO in modulation of neuropathic and inflammatory pain and support the hypothesis that activation of 5-HT5A receptors may inhibit the inhibitory effect of GABAergic interneurons on output neurons projecting to the PAG (GABAergic disinhibitory mechanisms), consequently activating the brainstem descending inhibitory system that depresses nociceptive transmission at the spinal cord level.

The norepinephrine (NE) system is involved in pathways that regulate morphine addiction. Here, we investigated the role of α1 adrenoceptor in the ventrolateral orbital cortex (VLO) of rats with repeated morphine treatment and underlying molecular mechanisms. The rewarding properties of morphine were assessed by the conditioned place preference (CPP) paradigm. Prazosin, an α1 adrenoceptor antagonist, was microinjected into the VLO. The expression of α1 adrenoceptor, p-CaMKII/CaMKII, CRTC1, BDNF and PSD95 in the VLO were determined by immunohistochemistry or western blotting. Neurotransmitter NE in the VLO and inflammatory factors in serum were detected separately through high-performance liquid chromatography and enzyme-linked immunosorbent assay. Our experimental results showed that repeated morphine administration induced stable CPP and prazosin promoted the morphine-induced CPP. Microinjection of prazosin in the VLO not only blocked the activity of α1 adrenoceptor, decreased CaMKII phosphorylation and CRTC1, which eventually resulted in a regression of synaptic plasticity-related proteins, but also was accompanied by significantly decreasing of NE in the VLO and increasing of inflammatory cytokines in peripheral blood. These findings suggested that prazosin potentiates the addictive effects of morphine. The effect of increased CPP through reducing α1 adrenoceptor and NE was associated with the CaMKII-CRTC1 pathway and synaptic plasticity-related proteins in the VLO and inflammatory cytokines in the peripheral blood. The NE system may therefore be an underlying therapeutic target in morphine addiction. Additionally, we believe that the clinical use of prazosin in hypertensive patients with morphine abuse may be a potential risk because of its reinforcing effect on addiction.

The 5-hydroxytryptamine 7 receptor (5-HT7R) is one of the most recently cloned serotonin receptors which have been implicated in many physiological and pathological processes including drug addiction. Behavioral sensitization is the progressive process during which re-exposure to drugs intensified the behavioral and neurochemical responses to drugs. Our previous study has demonstrated that the ventrolateral orbital cortex (VLO) is critical for morphine-induced reinforcing effect. The aim of the present study was to investigate the effect of 5-HT7Rs in the VLO on morphine-induced behavioral sensitization and their underlying molecular mechanisms. Our results showed that a single injection of morphine, followed by a low challenge dose could induce behavioral sensitization. Microinjection of the selective 5-HT7R agonist AS-19 into the VLO during the development phase significantly increased morphine-induced hyperactivity. Microinjection of the 5-HT7R antagonist SB-269970 suppressed acute morphine-induced hyperactivity and the induction of behavioral sensitization, but had no effect on the expression of behavioral sensitization. In addition, the phosphorylation of AKT (Ser 473) was increased during the expression phase of morphine-induced behavioral sensitization. Suppression of the induction phase could also block the increase of p-AKT (Ser 473). In conclusion, we demonstrated that 5-HT7Rs and p-AKT in the VLO at least partially contribute to morphine-induced behavioral sensitization.

Neurofilament light chain (NF-L) plays critical roles in synapses that are relevant to neuropsychiatric diseases. Despite postmortem evidence that NF-L is decreased in opiate abusers, its role and underlying mechanisms remain largely unknown. We found that the microinjection of the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) into the ventrolateral orbital cortex (VLO) attenuated chronic morphine-induced behavioral sensitization. The microinjection of TSA blocked the chronic morphine-induced decrease of NF-L. However, our chromatin immunoprecipitation (ChIP)-qPCR results indicated that this effect was not due to the acetylation of histone H3-Lysine 9 and 14 binding to the NF-L promotor. In line with the behavioral phenotype, the microinjection of TSA also blocked the chronic morphine-induced increase of p-ERK/p-CREB/p-NF-L. Finally, we compared chronic and acute morphine-induced behavioral sensitization. We found that although both chronic and acute morphine-induced behavioral sensitization were accompanied by an increase of p-CREB/p-NF-L, TSA exhibited opposing effects on behavioral phenotype and molecular changes at different addiction contexts. Thus, our findings revealed a novel role of NF-L in morphine-induced behavioral sensitization, and therefore provided some correlational evidence of the involvement of NF-L in opiate addiction.

Although many studies have indicated that orbitofrontal cortex plays an important role in the learning and retrieval of memory and subsequent decision-making, the role of ventrolateral orbital cortex (VLO) still remains unclear, especially related to fear and space.
Four separate cohorts of rats were used in this study. After sham surgery and electrical lesion of bilateral VLO, four cohorts received active avoidance test, passive avoidance test, Morris water maze and T maze separately.
Firstly, data shown that electrolytic lesions of bilateral VLO of Sprague-Dawley rats shortened the latency of rats to escape to darkroom in passive avoidance test. Besides, the damage of VLO also resulted in decrease of the number of active avoidance of rats from the third day during 5 consecutive days\' training in active avoidance test. What\'s more, the impairment of VLO significantly shortened the exploring time in the target quadrant of rats in Morris water maze. Furthermore, VLO-lesions group shown lower correct alternation percentage than sham group in T maze.
These results indicated that not only in the learning and retrieval of fear-related memory, VLO also plays an important role in the learning and retrieval of spatial-related memory guided by visual cues.

Previous studies have revealed that ventrolateral orbital cortex (VLO) may play an important role in the regulation of emotional behavior. However, it is not known what effect VLO damage will have on emotion regulation.
Data showed that damage of VLO increased the anxiety-like behavior in open field test and elevated plus maze, and decreased the depressive behavior in forced swimming test and learned helplessness test. Besides, the impulsive aggressive behaviors were also increased while the attack latency decreased after VLO lesion. What\'s more, damage of VLO decreased depressive behaviors induced by chronic unpredicted mild stress in rats.
These results suggest that the integrity of VLO plays an important role in emotional regulation, and the damage of VLO may inhibit the development of depression-like behavior.

Morphine addiction is categorized as a chronic recurrent brain disease which always results in mental disturbance, concomitant diseases and early death. Recent evidence suggested that Sirtuin 1 (SIRT1) played a crucial role in learning, memory and reward, nevertheless, its role in morphine addiction is still unclear. We explored whether SIRT1 in the ventrolateral orbital cortex (VLO) is associated with morphine addiction and its potential mechanism. We applied the morphine-induced behavioral sensitization paradigm to investigate whether microinjection of EX527, a SIRT1 inhibitor, into the VLO could affect the rat behaviors. Furthermore, we focused on the expression of extracellular signal-regulated protein kinases (ERK) and brain-derived neurotrophic factor (BDNF), potential downstream targets of SIRT1. Microinjecting EX527 into the VLO significantly suppressed morphine-induced behavioral sensitization. We found that the expression of SIRT1, phosphorylated ERK (p-ERK) and BDNF in the VLO were markedly up-regulated by morphine administrations in expression phase. These positive changes were significantly inhibited by microinjecting EX527 into the VLO. These results suggest that SIRT1 in the VLO may mediate morphine-induced behavioral sensitization and the overexpression of SIRT1, p-ERK and BDNF could be the potential mechanism. Taken together, the results of our research provide evidence to support that SIRT1 play an important role in morphine vulnerability and microinjecting EX527 into the VLO could significantly suppress morphine addiction in rats.

The nodose and jugular vagal ganglia supply sensory innervation to the airways and lungs. Jugular vagal airway sensory neurons wire into a brainstem circuit with ascending projections into the submedius thalamic nucleus (SubM) and ventrolateral orbital cortex (VLO), regions known to regulate the endogenous analgesia system. Here we investigate whether the SubM-VLO circuit exerts descending regulation over airway vagal reflexes in male and female rats using a range of neuroanatomical tracing, reflex physiology, and chemogenetic techniques. Anterograde and retrograde neuroanatomical tracing confirmed the connectivity of the SubM and VLO. Laryngeal stimulation in anesthetized rats reduced respiration, a reflex that was potently inhibited by activation of SubM. Conversely, inhibition of SubM potentiated laryngeal reflex responses, while prior lesions of VLO abolished the effects of SubM stimulation. In conscious rats, selective chemogenetic activation of SubM neurons specifically projecting to VLO significantly inhibited respiratory responses evoked by inhalation of the nociceptor stimulant capsaicin. Jugular vagal inputs to SubM via the medullary paratrigeminal nucleus were confirmed using anterograde transsynaptic conditional herpes viral tracing. Respiratory responses evoked by microinjections of capsaicin into the paratrigeminal nucleus were significantly attenuated by SubM stimulation, whereas those evoked via the nucleus of the solitary tract were unaltered. These data suggest that jugular vagal sensory pathways input to a nociceptive thalamocortical circuit capable of regulating jugular sensory processing in the medulla. This circuit organization suggests an intersection between vagal sensory pathways and the endogenous analgesia system, potentially important for understanding vagal sensory processing in health and mechanisms of hypersensitivity in disease.SIGNIFICANCE STATEMENT Jugular vagal sensory pathways are increasingly recognized for their important role in defensive respiratory responses evoked from the airways. Jugular ganglia neurons wire into a central circuit that is notable for overlapping with somatosensory processing networks in the brain rather than the viscerosensory circuits in receipt of inputs from the nodose vagal ganglia. Here we demonstrate a novel and functionally relevant example of intersection between vagal and somatosensory processing in the brain. The findings of the study offer new insights into interactions between vagal and spinal sensory processing, including the medullary targets of the endogenous analgesia system, and offer new insights into the central processes involved in airway defense in health and disease.

Mechanical allodynia, characterized by a painful sensation induced by innocuous stimuli, is thought to be caused by disruption in pain-related regions. Identification and reversal of this pathologic neuroadaptation are therefore beneficial for clinical treatment. Previous evidence suggests that 5-HT6 receptors in the ventrolateral orbital cortex (VLO) are involved in neuropathic pain, but their function is poorly understood. The aim of the present study is to unveil the role of 5-HT6 receptors in the VLO and the underlying mechanisms in pain modulation. Here, by using the spared nerve injury (SNI) pain model, first, we report that 5-HT6 receptor protein decreased in the contralateral VLO compared with the ipsilateral VLO in rats with allodynia. Second, microinjection of the selective 5-HT6 receptor agonists EMD-386088 and WAY-208466 into the contralateral VLO consistently and significantly depressed allodynia. Third, microinjection of the selective antagonist SB-258585 blocked the agonist-induced anti-allodynic effect, while the antagonist applied alone to the VLO had no effect. Furthermore, the anti-nociceptive effect of EMD-386088 on neuropathic pain was prevented by the adenylate cyclase (AC) inhibitor SQ-22536, and protein kinase A (PKA) inhibitor H89, suggesting that AC/PKA signaling might underlie the antinociception of agonists. Finally, the 5-HT6 receptors were found to be colocalized with a glutamate transporter (EAAC1) by immunofluorescent staining, and the glutamate receptor antagonist kynurenic acid was found to completely block antinociception. These findings indicated that the antinociceptive effect of 5-HT6 receptor agonists might occur via interaction with the glutamatergic system. Altogether, the agonists activated 5-HT6 receptors present in the glutamatergic neurons in the VLO to facilitate the AC/PKA cascade, which subsequently might evoke glutamate release, thus depressing allodynia. These findings suggest a potential therapeutic role of 5-HT6 receptor agonists in treating neuropathic pain.