Phosphorylation of Neurofilament Light Chain in the VLO Is Correlated with Morphine-Induced Behavioral Sensitization in Rats.

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.

Activation of α1 adrenoceptors in ventrolateral orbital cortex attenuates allodynia induced by spared nerve injury in rats.

Recent studies have demonstrated that noradrenaline acting in the ventrolateral orbital cortex (VLO) can potentially reduce allodynia induced by spared nerve injury (SNI), and this effect is mediated by α2 adrenoceptor. The present study examined the effect of the α1 adrenoceptors in the VLO on allodynia induced by SNI in the rats. The mechanical paw withdrawal threshold (PWT) was measured using von-Frey filaments. Microinjection of selective α1 adrenoceptor agonist methoxamine (20, 50, 100 µg in 0.5 µl) into the VLO, contralateral to the site of nerve injury, increased PWT in a dose-dependent manner. This effect was antagonized by pre-microinjection of the selective α1 adrenoceptor antagonist benoxathian into the same VLO site, and blocked by electrolytic lesion of the ventrolateral periaqueductal gray (PAG). Furthermore, pre-administration of non-selective glutamate receptor antagonist kynurenic acid, phospholipase C (PLC) inhibitor U73122, and protein kinase C (PKC) inhibitor chelerythrine to the VLO also blocked methoxamine-induced inhibition of allodynia. These results suggest that activation of α1 adrenoceptors in the VLO can potentially reduce allodynia induced by SNI. This effect may be direct excitation of the VLO neurons, via PLC-PKC signaling pathway, projecting to the PAG or facilitating glutamate release and then indirectly exciting the VLO output neurons projecting to the PAG, leading to activation of the PAG-brainstem descending inhibitory system which depresses the nociceptive transmission at the spinal cord level.

Microinjection of histone deacetylase inhibitor into the ventrolateral orbital cortex potentiates morphine induced behavioral sensitization.

Accumulating evidence indicates that epigenetic regulation, such as changes in histone modification in reward-related brain regions, contributes to the memory formation of addiction to opiates and psychostimulants. Our recent results suggested that the ventrolateral orbital cortex (VLO) is involved in the memories of stress and drug addiction. Since addiction and stress memories share some common pathways, the present study was designed to investigate the role of histone deacetylase (HDAC) activity in the VLO during morphine induced-behavioral sensitization. Rats received a single exposure to morphine for establishing the behavioral sensitization model. The effect of HDAC activity in the VLO in morphine induced-behavioral sensitization was examined by microinjection of HDAC inhibitor Trichostatin A (TSA). Furthermore, the protein expression levels of extracellular signal-regulated kinase (ERK) and phosphorylated ERK (p-ERK), histone H3 lysine 9 acetylation (aceH3K9) and brain-derived neurotrophic factor (BDNF) in the VLO in morphine-induced behavioral sensitization were examined. The results showed that the bilateral VLO lesions suppressed the expression phase, but not the developmental phase of morphine-induced behavioral sensitization. Microinjection of TSA into the VLO significantly increased both the development and expression phases. Moreover, the protein levels of p-ERK, aceH3K9 and BDNF except ERK in the VLO were significantly upregulated in morphine-treated rats in the expression phase. These effects were further strengthened by intra-VLO injection of TSA. Our findings suggest that HDAC activity in the VLO could potentiate morphine-induced behavioral sensitization. The upregulated expression of p-ERK, aceH3K9 and BDNF in the VLO might be the underlying mechanism of histone acetylation enhancing the morphine-induced behavioral sensitization.

The α1 adrenoceptors in ventrolateral orbital cortex contribute to the expression of morphine-induced behavioral sensitization in rats.

The aim of the present study was to investigate the effect of microinjection of benoxathian, selective α1 adrenoceptor antagonist, into the ventrolateral orbital cortex (VLO) on morphine-induced behavioral sensitization and its underlying molecular mechanism in rats. A single morphine treatment protocol was used in establishing the behavioral sensitization model. The effect of bilateral intra-VLO benoxathian injection on locomotor activity was examined and the protein expression levels of α1 adrenoceptors and activation of extracellular signal-regulated kinase (ERK) in the VLO were detected after locomotor test. The results showed that a single injection of morphine could induce behavioral sensitization by a low challenge dosage of morphine after a 7-days drug free period. Benoxathian significantly suppressed the expression but not the development of morphine-induced behavioral sensitization. Morphine treatment significantly elicited ERK phosphorylation and downregulated the expression level of α1 adrenoceptors in the VLO. In addition, intra-VLO benoxathian injection enhanced the expression levels of α1 adrenoceptors and phosphorylated ERK. These results suggest that α1 adrenoceptors in the VLO are involved in regulating the expression of morphine-induced behavioral sensitization. The effect of decreased locomotor activity by blocking α1 adrenoceptors might be associated with activation of ERK in the VLO.

[The effect of Salvianolic Acid B on the Apoptosis of HUVECs Induced by Intermittent High Glucose].

Objective: To investigate the effect of Salvianolic acid B (Sal B) on the apoptosis of human umbilical vein endothelial cells (HUVECs) induced by intermittent high glucose and to explore the possible mechanisms. Methods: HUVECs were preincubated with Sal B for 24 h, followed by incubation with intermittent high glucose (IHG, 5.5 mmol/L 12 h, 33.3 mmol/L 12 h) for 72 h. The viability of the HUVECs was determined by MTT assay, and the cells apoptosis was measured flow cytometry, respectively. The levels of nitric oxide (NO), total antioxidant capacity (T-AOC), malondialdehyde (MDA), and Caspase-3 activity were determined by colorimetric method. Intracellular ROS was evaluated by fluorescent microscopy. The protein levels of NOX4, p-eNOS, BAX, and BCL-2 were determined by Western-blot. Results: Pretreatment with Sal B significantly ameliorated IHG-induced cells injury as was manifested by increased cell viability, up-regulated eNOS activation, and promoted the release of NO in HUVECs (P < 0.05 or P < 0.01). Sal B evidently suppressed IHG-induced cell apoptosis, down-regulated the expression of BAX protein and up-regulated the expression of BCL-2 protein. The activity of Capase-3 was also significantly reduced. Pre-incubation with Sal B led to a significant enhancement of antioxidant capacity and a reduction of NOX4 protein expression, accompanied by a remarkable decrease of intracellular ROS and MDA content (P < 0.05 or P < 0.01). Conclusion: Sal B is capable of suppressing IHG-induced injury and apoptosis in HUVECs, which might be attributed to the attenuation of oxidative stress, regulation of BCL-2/BAX protein expression, and subsequent suppression of Caspase-3 activity.