A dedicated hypothalamic oxytocin circuit controls aversive social learning.

To survive in a complex social group, one needs to know who to approach and, more importantly, who to avoid. In mice, a single defeat causes the losing mouse to stay away from the winner for weeks1. Here through a series of functional manipulation and recording experiments, we identify oxytocin neurons in the retrochiasmatic supraoptic nucleus (SOROXT) and oxytocin-receptor-expressing cells in the anterior subdivision of the ventromedial hypothalamus, ventrolateral part (aVMHvlOXTR) as a key circuit motif for defeat-induced social avoidance. Before defeat, aVMHvlOXTR cells minimally respond to aggressor cues. During defeat, aVMHvlOXTR cells are highly activated and, with the help of an exclusive oxytocin supply from the SOR, potentiate their responses to aggressor cues. After defeat, strong aggressor-induced aVMHvlOXTR cell activation drives the animal to avoid the aggressor and minimizes future defeat. Our study uncovers a neural process that supports rapid social learning caused by defeat and highlights the importance of the brain oxytocin system in social plasticity.

PGE2 Potentiates Orai1-Mediated Calcium Entry Contributing to Peripheral Sensitization.

Peripheral sensitization is one of the primary mechanisms underlying the pathogenesis of chronic pain. However, candidate molecules involved in peripheral sensitization remain incompletely understood. We have shown that store-operated calcium channels (SOCs) are expressed in the dorsal root ganglion (DRG) neurons. Whether SOCs contribute to peripheral sensitization associated with chronic inflammatory pain is elusive. Here we report that global or conditional deletion of Orai1 attenuates Complete Freund's adjuvant (CFA)-induced pain hypersensitivity in both male and female mice. To further establish the role of Orai1 in inflammatory pain, we performed calcium imaging and patch-clamp recordings in wild-type (WT) and Orai1 knockout (KO) DRG neurons. We found that SOC function was significantly enhanced in WT but not in Orai1 KO DRG neurons from CFA- and carrageenan-injected mice. Interestingly, the Orai1 protein level in L3/4 DRGs was not altered under inflammatory conditions. To understand how Orai1 is modulated under inflammatory pain conditions, prostaglandin E2 (PGE2) was used to sensitize DRG neurons. PGE2-induced increase in neuronal excitability and pain hypersensitivity was significantly reduced in Orai1 KO mice. PGE2-induced potentiation of SOC entry (SOCE) was observed in WT, but not in Orai1 KO DRG neurons. This effect was attenuated by a PGE2 receptor 1 (EP1) antagonist and mimicked by an EP1 agonist. Inhibition of Gq/11, PKC, or ERK abolished PGE2-induced SOCE increase, indicating PGE2-induced SOCE enhancement is mediated by EP1-mediated downstream cascade. These findings demonstrate that Orai1 plays an important role in peripheral sensitization. Our study also provides new insight into molecular mechanisms underlying PGE2-induced modulation of inflammatory pain.Significance Statement Store-operated calcium channel (SOC) Orai1 is expressed and functional in dorsal root ganglion (DRG) neurons. Whether Orai1 contributes to peripheral sensitization is unclear. The present study demonstrates that Orai1-mediated SOC function is enhanced in DRG neurons under inflammatory conditions. Global and conditional deletion of Orai1 attenuates complete Freund's adjuvant (CFA)-induced pain hypersensitivity. We also demonstrate that prostaglandin E2 (PGE2) potentiates SOC function in DRG neurons through EP1-mediated signaling pathway. Importantly, we have found that Orai1 deficiency diminishes PGE2-induced SOC function increase and reduces PGE2-induced increase in neuronal excitability and pain hypersensitivity. These findings suggest that Orai1 plays an important role in peripheral sensitization associated with inflammatory pain. Our study reveals a novel mechanism underlying PGE2/EP1-induced peripheral sensitization. Orai1 may serve as a potential target for pathological pain.

Choroidal circulation disturbance is an initial factor in outer retinal degeneration in rats under simulated weightlessness.

Objective: Microgravity contributes to ocular injury yet the underlying mechanism remains unclear. This study aims to elucidate the mechanism behind choroidal circulation disorder and outer retinal degeneration in rats with simulated weightlessness. Methods: Optical coherence tomography angiography (OCTA) was used to evaluate choroidal circulation and retinal morphological alterations in rats with weightlessness simulation. Electroretinogram and transmission electron microscopy were used to examine the ultrastructure and function of the choroid and outer retina. Furthermore, histological and terminal deoxynucleotidyl transferase deoxyuridine dUTP nick-end labeling (TUNEL) staining was used to monitor retinal morphology. Western blotting was performed to analyze the expressions of blood-retinal outer barrier function-related proteins (Cx43, ZO-1, and occludin). Results: The choroidal thickening was observed from the fourth week of simulated weightlessness (p < 0.05), and choroidal capillary density started to decline by the fifth week (p < 0.05). Transmission electron microscopy revealed that the choroidal vessels were open and operating well by the fourth week. However, most of the mitochondria within the vascular endothelium underwent mild swelling, and by the fifth week, the choroidal vessels had various degrees of erythrocyte aggregation, mitochondrial swelling, and apoptosis. Additionally, ERG demonstrated a decline in retinal function beginning in the fifth week (p < 0.05). TUNEL staining revealed a significantly higher apoptotic index in the outer nuclear layer of the retina (p < 0.05). At the sixth week weeks of simulated weightlessness, OCTA and hematoxylin and eosin (HE) staining of retinal sections revealed that the outer nuclear layer of the retina started to become thin (p < 0.05). Results from western blotting revealed that Cx43, ZO-1, and occludin exhibited decreased expression (p < 0.05). Conclusion: Based on our findings in a rat model of simulated weightlessness, choroidal circulation disturbance induced by choroidal congestion is the initial cause of outer retinal degeneration. Blood-retinal barrier disruption is significant in this process.

A hypothalamic pathway that suppresses aggression toward superior opponents.

Aggression is costly and requires tight regulation. Here we identify the projection from estrogen receptor alpha-expressing cells in the caudal part of the medial preoptic area (cMPOAEsr1) to the ventrolateral part of the ventromedial hypothalamus (VMHvl) as an essential pathway for modulating aggression in male mice. cMPOAEsr1 cells increase activity mainly during male-male interaction, which differs from the female-biased response pattern of rostral MPOAEsr1 (rMPOAEsr1) cells. Notably, cMPOAEsr1 cell responses to male opponents correlated with the opponents' fighting capability, which mice could estimate based on physical traits or learn through physical combats. Inactivating the cMPOAEsr1-VMHvl pathway increased aggression, whereas activating the pathway suppressed natural intermale aggression. Thus, cMPOAEsr1 is a key population for encoding opponents' fighting capability-information that could be used to prevent animals from engaging in disadvantageous conflicts with superior opponents by suppressing the activity of VMHvl cells essential for attack behaviors.

Necroptosis plays a crucial role in the exacerbation of retinal injury after blunt ocular trauma.

Retinal injury after blunt ocular trauma may directly affect prognosis and lead to vision loss. To investigate the pathological changes and molecular mechanisms involved in retinal injury after blunt ocular trauma, we established a weight drop injury model of blunt ocular trauma in male Beagle dogs. Hematoxylin-eosin staining, immunofluorescence staining, western blotting, and TUNEL assays were performed to investigate retinal injury within 14 days after blunt ocular trauma. Compared with the control group, the thicknesses of the inner and outer nuclear layers, as well as the number of retinal ganglion cells, gradually decreased within 14 days after injury. The number of bipolar cells in the inner nuclear layer began to decrease 1 day after injury, while the numbers of cholinergic and amacrine cells in the inner nuclear layer did not decrease until 7 days after injury. Moreover, retinal cell necroptosis increased with time after injury; it progressed from the ganglion cell layer to the outer nuclear layer. Visual electrophysiological findings indicated that visual impairment began on the first day after injury and worsened over time. Additionally, blunt ocular trauma induced nerve regeneration and Müller glial hyperplasia; it also resulted in the recruitment of microglia to the retina and polarization of those microglia to the M1 phenotype. These findings suggest that necroptosis plays an important role in exacerbating retinal injury after blunt ocular trauma via gliosis and neuroinflammation. Such a role has important implications for the development of therapeutic strategies.

Frequency cumulative effect of subthreshold energy laser-activated remote phosphors irradiation on visual function in guinea pigs.

AIM: To explore the effects of laser-activated remote phosphors (LARP) on visual function in guinea pigs. METHODS: Electroretinogram (ERG) of guinea pigs were observed after LARP irradiation at different frequencies and irradiation times. We evaluated the expression of rhodopsin, ß-catenin, connexin36, calretinin, and calbindin in the retina of guinea pigs and measured the density of photoreceptor cells after high-frequency LARP irradiation. RESULTS: After LARP irradiation, the ERG results showed that the amplitude of the dark-adapted 3.0 b-wave of the model eye was lower than that of the control eye after high-frequency irradiation (P<0.05). The expression of rhodopsin, ß-catenin, connexin36, calretinin, and calbindin in the retina of guinea pig declined. CONCLUSION: There is frequency cumulative damage effect on the retina that relates to LARP illumination frequency. This has significance for staff visual protection policies under LARP lighting conditions.

ALDH2/SIRT1 Contributes to Type 1 and Type 2 Diabetes-Induced Retinopathy through Depressing Oxidative Stress.

Clinical observations found vision-threatening diabetic retinopathy (DR) occurs in both type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM) patients, but T1DM may perform more progressive retinal abnormalities at the same diabetic duration with or without clinical retinopathy. In the present study, T1DM and T2DM patients without manifestations of DR were included in our preliminary clinical retrospective observation study to investigate the differentiated retinal function at the preclinical stage. Then, T1DM and T2DM rat models with 12-week diabetic duration were constructed to explore the potential mechanism of the discrepancy in retinal disorders. Our data demonstrated T1DM patients presented a poor retinal function, a higher allele frequency for ALDH2GA/AA, and a depressed aldehyde dehydrogenase 2 (ALDH2) activity and silent information regulator 1 (SIRT1) level, compared to T2DM individuals. In line with this, higher amplitudes of neurovascular function-related waves of electroretinograms were found in T2DM rats. Furthermore, the retinal outer nuclear layers were reduced in T1DM rats. The levels of retinal oxidative stress biomarkers including total reactive oxygen species, NADPH oxidase 4 and mitochondrial DNA damage, and inflammatory indicators covering inducible/endothelial nitric acid synthase ratio, interleukin-1, and interleukin-6 were obviously elevated. Notably, the level of retinal ALDH2 and SIRT1 in T1DM rats was significantly diminished, while the expression of neovascularization factors was dramatically enhanced compared to T2DM. Together, our data indicated that the ALDH2/SIRT1 deficiency resulted in prominent oxidative stress and was in association with DR progression. Moreover, a differentiating ALDH2/SIRT1 expression may be responsible for the dissimilar severity of DR pathological processes in chronic inflammatory-related T1DM and T2DM.

Therapeutic effect of a traditional Chinese medicine formulation on experimental choroidal neovascularization in mouse.

AIM: To investigate therapeutic effects of traditional Chinese medicine formulations, Hexuemingmu (HXMM) on laser-induced choroidal neovascularization (CNV) and follow-up effect in mice. METHODS: C57BL/6 mice of 8-week-old were used and CNV was induced with 577 nm laser photocoagulation. Animals were randomly divided into groups and different doses of HXMM were administered daily. One, four, and eight weeks after the intervention, the electroretinogram (ERG), fundus fluorescence angiography, choroidal flat mount and immunofluorescence staining were preformed to evaluate the function and CNV formation. The expression levels of angiogenic proteins were determined by Western blotting and immunofluorescence staining. An analysis of variance and Kruskal-Wallis test were used to test the differences among the groups. RESULTS: The results showed that HXMM effectively increased amplitude of ERG of mice (P<0.05), alleviated fundus CNV leakage (P<0.05), and reduced the area of neovascularization and the expression of angiogenic proteins (P<0.05) after laser-induced CNV. CONCLUSION: HXMM can protect the retinal function of mice after laser-induced CNV, and inhibit the CNV development.

Retinal neovascularization induced by mutant Vldlr gene inhibited in an inherited retinitis pigmentosa mouse model: an in-vivo study.

AIM: To explore whether the retinal neovascularization (NV) in a genetic mutant mice model could be ameliorated in an inherited retinitis pigmentosa (RP) mouse, which would help to elucidate the possible mechanism and prevention of retinal NV diseases in clinic. METHODS: The Vldlr -/- mice, the genetic mutant mouse model of retinal NV caused by the homozygous mutation of Vldlr gene, with the rd1 mice, the inherited RP mouse caused by homozygous mutation of Pde6b gene were bred. Intercrossing of the above two mice led to the birth of the F1 hybrids, further inbreeding of which gave birth to the F2 offspring. The ocular genotypes and phenotypes of the mice from all generations were examined, with the F2 offspring grouped according to the genotypes. RESULTS: The rd1 mice exhibited the RP phenotype of outer retinal degeneration and loss of retinal function. The Vldlr -/- mice exhibited the phenotype of retinal NV obviously shown by the fundus fluorescein angiography. The F1 hydrides, with the heterozygote genotype, exhibited no phenotypes of RP or retinal NV. The F2 offspring with homozygous genotypes were grouped into four subgroups. They were the F2-I mice with the wild-type Pde6b and Vldlr genes (Pde6b+/+ -Vldlr+/+ ), which had normal ocular phenotypes; the F2-II mice with homozygous mutant Vldlr gene (Pde6b+/+ -Vldlr-/- ), which exhibited the retinal NV phenotype; the F2-III mice with homozygous mutant Pde6b gene (Pde6b-/- -Vldlr+/+ ), which exhibited the RP phenotype. Specifically, the F2-IV mice with homozygous mutant Vldlr and Pde6b gene (Pde6b-/- -Vldlr-/- ) showed only the RP phenotype, without the signs of retinal NV. CONCLUSION: The retinal NV can be inhibited by the RP phenotype, which implies the role of a hyperoxic state in treating retinal NV diseases.

Neural circuits of social behaviors: Innate yet flexible.

Social behaviors, such as mating, fighting, and parenting, are fundamental for survival of any vertebrate species. All members of a species express social behaviors in a stereotypical and species-specific way without training because of developmentally hardwired neural circuits dedicated to these behaviors. Despite being innate, social behaviors are flexible. The readiness to interact with a social target or engage in specific social acts can vary widely based on reproductive state, social experience, and many other internal and external factors. Such high flexibility gives vertebrates the ability to release the relevant behavior at the right moment and toward the right target. This maximizes reproductive success while minimizing the cost and risk associated with behavioral expression. Decades of research have revealed the basic neural circuits underlying each innate social behavior. The neural mechanisms that support behavioral plasticity have also started to emerge. Here we provide an overview of these social behaviors and their underlying neural circuits and then discuss in detail recent findings regarding the neural processes that support the flexibility of innate social behaviors.

Posterior amygdala regulates sexual and aggressive behaviors in male mice.

Sexual and aggressive behaviors are fundamental to animal survival and reproduction. The medial preoptic nucleus (MPN) and ventrolateral part of the ventromedial hypothalamus (VMHvl) are essential regions for male sexual and aggressive behaviors, respectively. While key inhibitory inputs to the VMHvl and MPN have been identified, the extrahypothalamic excitatory inputs essential for social behaviors remain elusive. Here we identify estrogen receptor alpha (Esr1)-expressing cells in the posterior amygdala (PA) as a main source of excitatory inputs to the hypothalamus and key mediators for mating and fighting in male mice. We find two largely distinct PA subpopulations that differ in connectivity, gene expression, in vivo responses and social behavior relevance. MPN-projecting PAEsr1+ cells are activated during mating and are necessary and sufficient for male sexual behaviors, while VMHvl-projecting PAEsr1+ cells are excited during intermale aggression and promote attacks. These findings place the PA as a key node in both male aggression and reproduction circuits.

Hierarchical Representations of Aggression in a Hypothalamic-Midbrain Circuit.

Although the ventromedial hypothalamus ventrolateral area (VMHvl) is now well established as a critical locus for the generation of conspecific aggression, its role is complex, with neurons responding during multiple phases of social interactions with both males and females. It has been previously unclear how the brain uses this complex multidimensional signal and coordinates a discrete action: the attack. Here, we find a hypothalamic-midbrain circuit that represents hierarchically organized social signals during aggression. Optogenetic-assisted circuit mapping reveals a preferential projection from VMHvlvGlut2 to lPAGvGlut2 cells, and inactivation of downstream lPAGvGlut2 populations results in aggression-specific deficits. lPAG neurons are selective for attack action and exhibit short-latency, time-locked spiking relative to the activity of jaw muscles during biting. Last, we find that this projection conveys male-biased signals from the VMHvl to downstream lPAGvGlut2 neurons that are sensitive to features of ongoing activity, suggesting that action selectivity is generated by a combination of pre- and postsynaptic mechanisms.

Protection of retinal function and morphology in MNU-induced retinitis pigmentosa rats by ALDH2: an in-vivo study.

BACKGROUND: Retinitis pigmentosa (RP) is a kind of inherited retinal degenerative diseases characterized by the progressive loss of photoreceptors. RP has been a conundrum without satisfactory countermeasures in clinic until now. Acetaldehyde dehydrogenase 2 (ALDH2), a major enzyme involved in aldehyde detoxification, has been demonstrated to be beneficial for a growing number of human diseases, such as cardiovascular dysfunction, diabetes mellitus and neurodegeneration. However, its protective effect against RP remains unknown. Our study explored the impact of ALDH2 on retinal function and structure in N-methyl-N-nitrosourea (MNU)-induced RP rats. METHODS: Rats were gavaged with 5 mg/kg Alda-1, an ALDH2 agonist, 5 days before and 3 days after MNU administration. Assessments of retinal function and morphology as well as measurement of specific proteins expression level were conducted. RESULTS: Electroretinogram recordings showed that Alda-1 administration alleviated the decrease in amplitude caused by MNU, rendering protection of retinal function. Mitigation of photoreceptor degeneration in MNU-treated retinas was observed by optical coherence tomography and retinal histological examination. In addition, Western blotting results revealed that ALDH2 protein expression level was upregulatedwith increased expression of SIRT1 protein after the Alda-1 intervention. Besides, endoplasmic reticulum stress (ERS) was reduced according to the significant downregulation of GRP78 protein, while apoptosis was ameliorated as shown by the decreased expression of PARP1 protein. CONCLUSIONS: Together, our data demonstrated that ALDH2 could provide preservation of retinal function and morphology against MNU-induced RP, with the underlying mechanism at least partly related to the modulation of SIRT1, ERS and apoptosis.

ALDH2 protects naturally aged mouse retina via inhibiting oxidative stress-related apoptosis and enhancing unfolded protein response in endoplasmic reticulum.

During the process of aging, the retina exhibits chronic oxidative stress (OS) damage. Our preliminary experiment showed that acetaldehyde dehydrogenase 2 (ALDH2) could alleviate retinal damage caused by OS. This study aimed to explore whether ALDH2 could inhibit mice retinal cell apoptosis and enhance the function of unfolded protein response in endoplasmic reticulum (UPRER) through reducing OS in aging process. Retinal function and structure in vivo and in vitro were examined in aged ALDH2+ overexpression mice and ALDH2 agonist Alda1-treated aged mice. Levels of ALDH2, endoplasmic reticulum stress (ERS), apoptosis and inflammatory cytokines were evaluated. Higher expression of ALDH2 was observed at the outer nuclear layer (ONL) and the inner nuclear layer (INL) in aged ALDH2+ overexpression and aged Alda1-treated mice. Moreover, aged ALDH2+ overexpression mice and aged Alda1-treated mice exhibited better retinal function and structure. Increased expression of glucose-regulated protein 78 (GRP78) and ERS-related protein phosphorylated eukaryotic initiation factor 2 (peIF2α) and decreased expression of apoptosis-related protein, including C/EBP homologous protein (CHOP), caspase12 and caspase9, and retinal inflammatory cytokines were detected in the retina of aged ALDH2+ overexpression mice and aged Alda1-treated mice. The expression of ALDH2 in the retina was decreased in aging process. ALDH2 could reduce retinal oxidative stress and apoptosis, strengthen UPRER during the aging process to improve retinal function and structure.

Orai1 Plays a Crucial Role in Central Sensitization by Modulating Neuronal Excitability.

Pathological pain is a common and debilitating condition that is often poorly managed. Central sensitization is an important mechanism underlying pathological pain. However, candidate molecules involved in central sensitization remain unclear. Store-operated calcium channels (SOCs) mediate important calcium signals in nonexcitable and excitable cells. SOCs have been implicated in a wide variety of human pathophysiological conditions, including immunodeficiency, occlusive vascular diseases, and cancer. However, the role of SOCs in CNS disorders has been relatively unexplored. Orai1, a key component of SOCs, is expressed in the human and rodent spinal cord dorsal horn, but its functional significance in dorsal horn neurons is poorly understood. Here we sought to explore a potential role of Orai1 in the modulation of neuronal excitability and A-type potassium channels involved in pain plasticity. Using both male and female Orai1 knock-out mice, we found that activation of Orai1 increased neuronal excitability and reduced A-type potassium channels via the protein kinase C-extracellular signal-regulated protein kinase (PKC-ERK) pathway in dorsal horn neurons. Orai1 deficiency significantly decreased acute pain induced by noxious stimuli, nearly eliminated the second phase of formalin-induced nociceptive response, markedly attenuated carrageenan-induced ipsilateral pain hypersensitivity and abolished carrageenan-induced contralateral mechanical allodynia. Consistently, carrageenan-induced increase in neuronal excitability was abolished in the dorsal horn from Orai1 mutant mice. These findings uncover a novel signaling pathway involved in the pain process and central sensitization. Our study also reveals a novel link among Orai1, ERK, A-type potassium channels, and neuronal excitability.SIGNIFICANCE STATEMENT Orai1 is a key component of store-operated calcium channels (SOCs) in many cell types. It has been implicated in such pathological conditions as immunodeficiency, autoimmunity, and cancer. However, the role of Orai1 in CNS disorders remains poorly understood. The functional significance of Orai1 in neurons is elusive. Here we demonstrate that activation of Orai1 modulates neuronal excitability and Kv4-containing A-type potassium channels via the protein kinase C-extracellular signal-regulated protein kinase (PKC-ERK) pathway. Genetic knock-out of Orai1 nearly eliminates the second phase of formalin-induced pain and markedly attenuates carrageenan-induced pain hypersensitivity and neuronal excitability. These findings reveal a novel link between Orai1 and neuronal excitability and advance our understanding of central sensitization.

Astragaloside Alleviates Hepatic Fibrosis Function via PAR2 Signaling Pathway in Diabetic Rats.

BACKGROUND/AIMS: Astragaloside (AGS) extracted from radix astragalin (Huangqi) has been considered to be beneficial to liver diseases. In this study, we examined the role played by AGS in alleviating hepatic fibrosis function via protease-activated receptor-2 (PAR2) mechanisms. We hypothesized that AGS affects PAR2 signaling pathway thereby improving hepatic function in rats with hepatic fibrosis induced by carbon tetrachloride (CCl4). We further hypothesized that AGS attenuates impaired hepatic function evoked by CCl4 to a greater degree in diabetic animals. METHODS: ELISA and Western Blot analysis were used to examine PAR2 signaling pathway in diabetic CCl4-rats and non-diabetic CCl4-rats. RESULTS: AGS inhibited the protein expression of PAR2 and its downstream pathway PKA and PKCɛ in CCl4-rats. Notably, the effects of AGS were greater in CCl4-rats with diabetes. AGS also significantly attenuated the CCl4-induced upregulations of pro-inflammatory cytokines, namely interleukin-1ß, interleukin-6 and tumor necrosis factor-α accompanied with decreases of collagenic parameters such as hexadecenoic acid, laminin and hydroxyproline. Additionally, AGS improved the CCl4-induced exaggerations of liver index and functions including alanine aminotransferase, aspartate aminotransferase. Moreover, TGF-ß1, a marker of hepatic fibrosis, was increased in CCl4-rats and AGS inhibited increases in TGF-ß1 induced by CCl4. CONCLUSIONS: AGS alleviates hepatic fibrosis by inhibiting PAR2 signaling expression and its effects are largely enhanced in diabetic animals. Targeting one or more of these signaling molecules may present new opportunities for treatment and management of hepatic fibrosis; and results of our study are likely to shed light on strategies for application of AGS because it has potentially greater therapeutic effectiveness for hepatic fibrosis in diabetes.

Orai1 and Orai3 Mediate Store-Operated Calcium Entry Contributing to Neuronal Excitability in Dorsal Root Ganglion Neurons.

Store-operated calcium channels (SOCs) are highly calcium-selective channels that mediate calcium entry in various cell types. We have previously reported that intraplantar injection of YM-58483 (a SOC inhibitor) attenuates chronic pain. A previous study has reported that the function of SOCs in dorsal root ganglia (DRG) is enhanced after nerve injury, suggesting that SOCs may play a peripheral role in chronic pain. However, the expression, functional distribution and significance of the SOC family in DRG neurons remain elusive and the key components that mediate SOC entry (SOCE) are still controversial. Here, we demonstrated that the SOC family (STIM1, STIM2, Orai1, Orai2, and Orai3) was expressed in DRGs and STIM1 was mainly present in small- and medium-sized DRG neurons. Using confocal live cell imaging, Ca2+ imaging and electrophysiology techniques, we demonstrated that depletion of the endoplasmic reticulum Ca2+ stores induced STIM1 and STIM2 translocation, and that inhibition of STIM1 or blockage of Orai channels with pharmacological tools attenuated SOCE and SOC currents. Using the small inhibitory RNA knockdown approach, we identified STIM1, STIM2, Orai1, and Orai3 as the key components of SOCs mediating SOCE in DRG neurons. Importantly, activation of SOCs by thapsigargin induced plasma membrane depolarization and increased neuronal excitability, which were completely abolished by inhibition of SOCs or double knockdown of Orai1 and Orai3. Our findings suggest that SOCs exert an excitatory action in DRG neurons and provide a potential peripheral mechanism for modulation of pain hypersensitivity by SOC inhibition.

[Ocular pharmacokinetics of puerarin in anesthetic rabbits by microdialysis].

OBJECTIVE: To establish the model of microdialysis, and study the ocular pharmacokinetics of puerarin in anesthetic rabbits. METHOD: Implanted the probe into anterior chamber of anesthetic rabbit by surgery. After balanced for 2 h, 1% puerarin eye drop (100 microL) was applied into the cul-de-sac with micropipette. Immediately the dialysate was collected at different time and detected by HPLC with the detection wavelength of 249 nm. The mobile phase was methanol and 0.1% citric acid solution (30:70); the flow rate was 1.0 mL x min(-1). RESULT: After the administration, puerarin can be absorbed into aqueous humor quickly. The peak concentration of puerarin appeared at about 1 h and then reduced gradually. The peak concentration(C(max)) is (2.52 +/- 0.31) mg x L(-1). The other lower peak was shown at 3.5 h during the eliminate phase. This might be attributed to the inhibition of aqueous humor production by the puerarin and resulted in a high drug concentration. The area under concentration-time curve (AUC(0-t)) is (5.04 +/- 0.21) mg x h x L(-1) and the eliminate half life (t1/2) is (0.38 +/- 0.13) h. CONCLUSION: The microdialysis technique can be used to detect the ocular pharmacokinetics of puerarin, and support the valuable pharmacokinetics parameter for the clinical applications of puerarin eye drop.