Calcification segmentation based on a different scales superpixels saliency detection algorithm.

Accurate detection of breast tumor calcifications is of great significance in assisting doctors' diagnosis to improve the accuracy of breast cancer early detection. In this article, a different scale of superpixels saliency detection algorithm is used to segment calcifications in breast tumor ultrasound images based on a simple linear iterative cluster. First, a multi-scale saliency segmentation algorithm was used to divide the tumor region of different sizes and weak calcification (Wca) was extracted according to uneven gray distribution and texture contrast between regions. Second, based on single-scale superpixel segmentation of the original image, the strong calcification extraction map was calculated by measuring gray value difference and calcification gray distance features. Finally, the final calcification extraction map was obtained by combining the strong and weak calcification extraction maps. The detection algorithm proposed in this article could effectively detect calcifications in breast ultrasound images.

[Multi-feature Extraction and Classification of Breast Tumor in Ultrasound Image].

OBJECTIVE: Feature extraction of breast tumors is very important in the breast tumor detection (benign and malignant) in ultrasound image. The traditional quantitative description of breast tumors has some shortcomings, such as inaccuracy. A simple and accurate feature extraction method has been studied. METHODS: In this paper, a new method of boundary feature extraction was proposed. Firstly, the shape histogram of ultrasound breast tumors was constructed. Secondly, the relevant boundary feature factors were calculated from a local point of view, including sum of maximum curvature, sum of maximum curvature and peak, sum of maximum curvature and standard deviation. Based on the boundary features, shape features and texture features, the linear support vector machine classifiers for benign and malignant breast tumor recognition was constructed. RESULTS: The accuracy of boundary features in the benign and malignant breast tumors classification was 82.69%. The accuracy of shape features was 73.08%. The accuracy of texture features was 63.46%. The classification accuracy of the three fusion features was 86.54%. CONCLUSIONS: The classification accuracy of boundary features was higher than that of texture features and shape features. The classification method based on multi-features has the highest accuracy and it describes the benign and malignant tumors from different angles. The research results have practical value.

DNA methylation-mediated Klotho silencing is an independent prognostic biomarker of head and neck squamous carcinoma.

PURPOSE: To study the prognostic value of klotho (KL) and its promoter DNA methylation in head and neck squamous cell carcinoma (HNSCC) and to assess their associations with the autophagy gene LC3 and the RNA transferase gene NSUN2. MATERIALS AND METHODS: Upper quartile normalized RNA-seq V2 RSEM values of KL mRNA and beta value for KL methylation were retrieved from The Cancer Genome Atlas HNSCC dataset. Kaplan-Meier survival curves were used to assess the associations of KL expression and methylation with patient survival; multivariate Cox proportional hazards regression models were used to estimate the HRs and their 95% CIs. RESULTS: There is a negative relationship between KL gene expression and its promoter DNA methylation in HNSCC. KL gene expression was positively correlated with overall survival, while KL methylation was inversely correlated with the overall survival of HNSCC patients. Furthermore, KL methylation was significantly associated with gender (P=0.012), tumor grade (P=0.0009) and tumor site (P<0.0001). Finally, HNSCC patients with high KL gene expression or low KL DNA methylation had high LC3 but low NSUN2. CONCLUSION: KL methylation silenced its gene expression in HNSCC. Low KL expression and high KL methylation can be potential biomarkers for worse prognosis in HNSCC. As the downstream targets, LC3 and NSUN2 could be responsible for the KL expression in HNSCC.

Activation of Serotonin 2C Receptors in Dopamine Neurons Inhibits Binge-like Eating in Mice.

BACKGROUND: Neural networks that regulate binge eating remain to be identified, and effective treatments for binge eating are limited. METHODS: We combined neuroanatomic, pharmacologic, electrophysiological, Cre-lox, and chemogenetic approaches to investigate the functions of 5-hydroxytryptamine (5-HT) 2C receptor (5-HT2CR) expressed by dopamine (DA) neurons in the regulation of binge-like eating behavior in mice. RESULTS: We showed that 5-HT stimulates DA neural activity through a 5-HT2CR-mediated mechanism, and activation of this midbrain 5-HT→DA neural circuit effectively inhibits binge-like eating behavior in mice. Notably, 5-HT medications, including fluoxetine, d-fenfluramine, and lorcaserin (a selective 5-HT2CR agonist), act on 5-HT2CRs expressed by DA neurons to inhibit binge-like eating in mice. CONCLUSIONS: We identified the 5-HT2CR population in DA neurons as one potential target for antibinge therapies, and provided preclinical evidence that 5-HT2CR agonists could be used to treat binge eating.

The ERα-PI3K Cascade in Proopiomelanocortin Progenitor Neurons Regulates Feeding and Glucose Balance in Female Mice.

Estrogens act upon estrogen receptor (ER)α to inhibit feeding and improve glucose homeostasis in female animals. However, the intracellular signals that mediate these estrogenic actions remain unknown. Here, we report that anorexigenic effects of estrogens are blunted in female mice that lack ERα specifically in proopiomelanocortin (POMC) progenitor neurons. These mutant mice also develop insulin resistance and are insensitive to the glucose-regulatory effects of estrogens. Moreover, we showed that propyl pyrazole triol (an ERα agonist) stimulates the phosphatidyl inositol 3-kinase (PI3K) pathway specifically in POMC progenitor neurons, and that blockade of PI3K attenuates propyl pyrazole triol-induced activation of POMC neurons. Finally, we show that effects of estrogens to inhibit food intake and to improve insulin sensitivity are significantly attenuated in female mice with PI3K genetically inhibited in POMC progenitor neurons. Together, our results indicate that an ERα-PI3K cascade in POMC progenitor neurons mediates estrogenic actions to suppress food intake and improve insulin sensitivity.

Estrogen receptor-α in medial amygdala neurons regulates body weight.

Estrogen receptor-α (ERα) activity in the brain prevents obesity in both males and females. However, the ERα-expressing neural populations that regulate body weight remain to be fully elucidated. Here we showed that single-minded-1 (SIM1) neurons in the medial amygdala (MeA) express abundant levels of ERα. Specific deletion of the gene encoding ERα (Esr1) from SIM1 neurons, which are mostly within the MeA, caused hypoactivity and obesity in both male and female mice fed with regular chow, increased susceptibility to diet-induced obesity (DIO) in males but not in females, and blunted the body weight-lowering effects of a glucagon-like peptide-1-estrogen (GLP-1-estrogen) conjugate. Furthermore, selective adeno-associated virus-mediated deletion of Esr1 in the MeA of adult male mice produced a rapid body weight gain that was associated with remarkable reductions in physical activity but did not alter food intake. Conversely, overexpression of ERα in the MeA markedly reduced the severity of DIO in male mice. Finally, an ERα agonist depolarized MeA SIM1 neurons and increased their firing rate, and designer receptors exclusively activated by designer drug-mediated (DREADD-mediated) activation of these neurons increased physical activity in mice. Collectively, our results support a model where ERα signals activate MeA neurons to stimulate physical activity, which in turn prevents body weight gain.

Progress in the molecular understanding of central regulation of body weight by estrogens.

OBJECTIVE: Estrogens can act in the brain to prevent body weight gain. Tremendous research efforts have been focused on estrogen physiology in the brain in the context of body weight control; estrogen receptors and the related signals have been attractive targets for development of new obesity therapies. The objective is to review recent findings on these aspects. METHODS: Recent studies that used conventional and conditional knockout mouse strains to delineate the cellular and molecular mechanisms for the beneficial effects of estrogens on body weight balance are reviewed. Emerging genetic tools that could further benefit the field of estrogen research and a newly developed estrogen-based regimen that produces body weight-lowering benefits also are discussed. RESULTS: The body weight-lowering effects of estrogens are mediated by multiple forms of estrogen receptors in different brain regions through distinct but coordinated mechanisms. Both rapid signals and "classic" nuclear receptor actions of estrogen receptors appear to contribute to estrogenic regulation of body weight. CONCLUSIONS: Estrogen receptors and associated signal networks are potential targets for obesity treatment, and further investigations are warranted.

Regulatory effects of anandamide on intracellular Ca(2+) concentration increase in trigeminal ganglion neurons.

Activation of cannabinoid receptor type 1 on presynaptic neurons is postulated to suppress neurotransmission by decreasing Ca(2+) influx through high voltage-gated Ca(2+) channels. However, recent studies suggest that cannabinoids which activate cannabinoid receptor type 1 can increase neurotransmitter release by enhancing Ca(2+) influx in vitro. The aim of the present study was to investigate the modulation of intracellular Ca(2+) concentration by the cannabinoid receptor type 1 agonist anandamide, and its underlying mechanisms. Using whole cell voltage-clamp and calcium imaging in cultured trigeminal ganglion neurons, we found that anandamide directly caused Ca(2+) influx in a dose-dependent manner, which then triggered an increase of intracellular Ca(2+) concentration. The cyclic adenosine and guanosine monophosphate-dependent protein kinase systems, but not the protein kinase C system, were involved in the increased intracellular Ca(2+) concentration by anandamide. This result showed that anandamide increased intracellular Ca(2+) concentration and inhibited high voltage-gated Ca(2+) channels through different signal transduction pathways.

Estrogens stimulate serotonin neurons to inhibit binge-like eating in mice.

Binge eating afflicts approximately 5% of US adults, though effective treatments are limited. Here, we showed that estrogen replacement substantially suppresses binge-like eating behavior in ovariectomized female mice. Estrogen-dependent inhibition of binge-like eating was blocked in female mice specifically lacking estrogen receptor-α (ERα) in serotonin (5-HT) neurons in the dorsal raphe nuclei (DRN). Administration of a recently developed glucagon-like peptide-1-estrogen (GLP-1-estrogen) conjugate designed to deliver estrogen to GLP1 receptor-enhanced regions effectively targeted bioactive estrogens to the DRN and substantially suppressed binge-like eating in ovariectomized female mice. Administration of GLP-1 alone reduced binge-like eating, but not to the same extent as the GLP-1-estrogen conjugate. Administration of ERα-selective agonist propylpyrazole triol (PPT) to murine DRN 5-HT neurons activated these neurons in an ERα-dependent manner. PPT also inhibited a small conductance Ca2+-activated K+ (SK) current; blockade of the SK current prevented PPT-induced activation of DRN 5-HT neurons. Furthermore, local inhibition of the SK current in the DRN markedly suppressed binge-like eating in female mice. Together, our data indicate that estrogens act upon ERα to inhibit the SK current in DRN 5-HT neurons, thereby activating these neurons to suppress binge-like eating behavior and suggest ERα and/or SK current in DRN 5-HT neurons as potential targets for anti-binge therapies.

Channelrhodopsin-2-expressed dorsal root ganglion neurons activates calcium channel currents and increases action potential in spinal cord.

STUDY DESIGN: We used optogenetic techniques in spinal cord and dorsal root ganglion (DRG) neuron studies. OBJECTIVE: This study investigated changes in channelrhodopsin-2 (ChR2) expression in the spinal cord and DRG neurons using optogenetic techniques. The results show the possibility of using optogenetics to treat neuropathic pain. SUMMARY OF BACKGROUND DATA: Previous studies have shown that activated ChR2 induces an increase in DRG neuron action potential. METHODS: Western blot analysis was used to measure ChR2 protein levels in the spinal cord and DRG neurons or rats intrathecally injected with ChR2 lentivirus. Electrophysiology recording was used to detect differences in action potential levels in the spinal cord and calcium channel currents in the DRG neurons. RESULTS: Our studies showed that ChR2 expression increased the action potential in the spinal cord and increased calcium channel currents in DRG neurons. CONCLUSION: We successfully expressed the ChR2 protein in the spinal cord and DRG neurons. We also found that ChR2 increased the action potential in the spinal cord and activated the calcium channel in DRG neurons. These findings support the research possibilities of using optogenetic studies to improve treatment for neuropathic pain. LEVEL OF EVIDENCE: N/A.

Increased heat shock transcription factor 1 in the cerebellum reverses the deficiency of Purkinje cells in Alzheimer's disease.

Alzheimer's disease (AD) is one of the most debilitating neurodegenerative nerve diseases, seriously affecting one's ability to carry out daily activities. AD is both progressive and incurable, but molecular studies have begun to shed light on the mechanisms that underlie it. Immunochemical staining showed that cell bodies of Purkinje cells in the cerebellum were significantly reduced in AD rats compared with normal rats. Heat shock protein 70 (HSP70) was found to prevent polyglutamine aggregation in Huntington's disease and spinocerebellar ataxias (SCAs) and to relieve symptoms in SCAs and Parkinson's disease. Recently, AD-related phenotypes were found to be suppressed in HSP70 transgenic rats. However, the effects of other HSPs and the mechanisms of HSP-triggered changes in AD are unknown. In this study, we found that expression levels of HSP60, -70, and -90 were downregulated in the cerebella of rats with AD. Furthermore, heat shock factor 1 (HSF1), a key transcription factor for the expression of HSP genes, was found to be greatly decreased in the cerebella of AD rats. Even more interesting, injection of lentivirus vector-HSF1 into the cerebella of AD rats significantly increased HSF1 and HSP expression levels and induced an increase in the number of Purkinje cell bodies. Our findings provide novel evidence that low expression of HSPs in AD rats is dependent on the low expression of HSF1, and increased expression of HSF1 contributes to the reversal of cerebellar Purkinje cell deficiency in AD. Therefore, increasing HSF1 expression is a potential new strategy for the treatment of AD.

Complex regulation of capsaicin on intracellular second messengers by calcium dependent and independent mechanisms in primary sensory neurons.

Intracellular second messengers play an important role in capsaicin- and analogous-induced sensitization and desensitization in pain. Fluorescence Ca²âº imaging, enzyme immunoassay and PKC assay kit were used to determine a novel mechanism of different Ca²âº dependency in the signal transduction of capsaicin-induced desensitization. On the average, capsaicin increased cAMP, cGMP concentration and SP release in bell-shaped concentration-dependent manner, with the maximal responses at concentrations around 1 µM, suggesting acute desensitization of TRPV1 receptor activation. Capsaicin-induced intracellular Ca²âº concentration ([Ca²âº](i)) increase depended on extracellular Ca²âº influx as an initial trigger. The Ca²âº influx by capsaicin increased PKC activation and SP release. These increases were completely abolished in Ca²âº-free solution, suggesting that the modulation of capsaicin on PKC and SP are Ca²âº-dependent. Interestingly, the maximal cAMP increase by TRPV1 activation was not blocked Ca²âº removal, suggesting at least in part a Ca²âº-independent pathway is involved. Further study showed that cAMP increase was totally abolished by G-protein and adenylate cyclase (AC) antagonist, suggesting a G-protein-dependent pathway in cAMP increase. However, SP release was blocked by inhibiting PKC, but not G-protein or AC, suggesting a G-protein independent pathway in SP release. These results suggest that both Ca²âº-dependent and independent mechanisms are involved in the regulation of capsaicin on second messengers systems, which could be a novel mechanism underlying distinct desensitization of capsaicin and might provide additional opportunities in the development of effective analgesics in pain treatment.

Nerve injury increases brain-derived neurotrophic factor levels to suppress BK channel activity in primary sensory neurons.

Abnormal hyperexcitability of primary sensory neurons contributes to neuropathic pain development after nerve injury. Nerve injury profoundly reduces the expression of big conductance Ca(2+) -activated K(+) (BK) channels in the dorsal root ganglion (DRG). However, little is known about how nerve injury affects BK channel activity in DRG neurons. In this study, we determined the changes in BK channel activity in DRG neurons in a rat model of neuropathic pain and the contribution of brain-derived neurotrophic factor (BDNF) to reduced BK channel activity. The BK channel activity was present predominantly in small and medium DRG neurons, and ligation of L5 and L6 spinal nerves profoundly decreased the BK current density in these neurons. Blocking BK channels significantly increased neuronal excitability in sham control, but not in nerve-injured, rats. The BDNF concentration in the DRG was significantly greater in nerve-injured rats than in control rats. BDNF treatment largely reduced BK currents in DRG neurons in control rats, which was blocked by either anti-BDNF antibody or K252a, a Trk receptor inhibitor. Furthermore, either anti-BDNF antibody or K252a reversed reduction in BK currents in injured DRG neurons. BDNF treatment reduced the mRNA levels of BKα1 subunit in DRG neurons, and anti-BDNF antibody attenuated the reduction in the BKα1 mRNA level in injured DRG neurons. These findings suggest that nerve injury primarily diminishes the BK channel activity in small and medium DRG neurons. Increased BDNF levels contribute to reduced BK channel activity in DRG neurons through epigenetic and transcriptional mechanisms in neuropathic pain.

Cannabinoid WIN 55,212-2 inhibits TRPV1 in trigeminal ganglion neurons via PKA and PKC pathways.

Although the inhibitory effect of cannabinoids on transient receptor potential vanilloid 1 (TRPV1) channel may explain the efficacy of peripheral cannabinoids in antihyperalgesia and antinociceptive actions, the mechanism for cannabinoid-induced inhibition of TRPV1 in primary sensory neurons is not understood. Therefore, we explored how WIN55,212-2 (WIN, a synthetic cannabinoid) inhibited TRPV1 in rat trigeminal ganglion neurons. A "bell"-shaped concentration-dependent curve was obtained from the effects of WIN on TRPV1 channel. The maximal inhibition on capsaicin-induced current (I (cap)) by WIN was at a concentration of 10(-9) M, and at this concentration I (cap) was reduced by 95 ± 1.6%. When the concentration of WIN was at 10(-6) M, it displayed a stimulatory effect on I (cap). In this study, several intracellular signaling transduction pathways were tested to study whether they were involved in the inhibitory effects of WIN on I (cap). We found that the inhibitory effect of WIN on I (cap) was completely reversed by PKA antagonists H-89 and KT5720 as well as by PKC antagonists BIM and staurosporine. It was also found that the inhibitory effect was partly reversed by PKG antagonist PKGi, while G-protein antagonist GDP-ßs/pertussis toxin (PTX) and PLC antagonist U-73122 had no effect on the inhibitory effect of WIN on I(cap). These results suggest that several intracellular signaling transduction pathways including PKA and PKC systems underlie the inhibitory effects of WIN on I (cap); however, G protein-coupled receptors CB1 or CB2 were not involved.

Up-regulation of Cavß3 subunit in primary sensory neurons increases voltage-activated Ca2+ channel activity and nociceptive input in neuropathic pain.

High voltage-activated calcium channels (HVACCs) are essential for synaptic and nociceptive transmission. Although blocking HVACCs can effectively reduce pain, this treatment strategy is associated with intolerable adverse effects. Neuronal HVACCs are typically composed of α(1), ß (Cavß), and α(2)δ subunits. The Cavß subunit plays a crucial role in the membrane expression and gating properties of the pore-forming α(1) subunit. However, little is known about how nerve injury affects the expression and function of Cavß subunits in primary sensory neurons. In this study, we found that Cavß(3) and Cavß(4) are the most prominent subtypes expressed in the rat dorsal root ganglion (DRG) and dorsal spinal cord. Spinal nerve ligation (SNL) in rats significantly increased mRNA and protein levels of the Cavß(3), but not Cavß(4), subunit in the DRG. SNL also significantly increased HVACC currents in small DRG neurons and monosynaptic excitatory postsynaptic currents of spinal dorsal horn neurons evoked from the dorsal root. Intrathecal injection of Cavß(3)-specific siRNA significantly reduced HVACC currents in small DRG neurons and the amplitude of monosynaptic excitatory postsynaptic currents of dorsal horn neurons in SNL rats. Furthermore, intrathecal treatment with Cavß(3)-specific siRNA normalized mechanical hyperalgesia and tactile allodynia caused by SNL but had no significant effect on the normal nociceptive threshold. Our findings provide novel evidence that increased expression of the Cavß(3) subunit augments HVACC activity in primary sensory neurons and nociceptive input to dorsal horn neurons in neuropathic pain. Targeting the Cavß(3) subunit at the spinal level represents an effective strategy for treating neuropathic pain.

The modulation of the excitability of primary sensory neurons by Ca²âº-CaM-CaMKII pathway.

Ca(2+)-calmodulin (CaM) dependent protein kinase II (CaMKII) is an important intracellular signal transduction pathway. CaMKII is rich in the primary sensory neurons and specifically presents in the small- and medium-sized neurons. It remains unclear about the modulation on the excitability of primary sensory neurons by Ca(2+)-CaM-CaMKII pathway. By current clamp recording, we found that the excitability of capsaicin-sensitive small and medium trigeminal ganglion (TG) neurons was significantly reduced by a CaM specific antagonist (W-7) and a CaMKII antagonist (KN-93). The inhibition is represented as the reduction of numbers of action potential (AP), decrease of the amplitude of AP, increase of threshold, and prolongation of duration of AP. Consistently, by voltage clamp recording, we found that both voltage-gated sodium channels (VGSCs) and voltage-gated potassium channels (VGPCs) were inhibited by W-7 and KN-93 in the order of total sodium (Na(+)) current (INa-T) > sustained potassium (K(+)) current (IK) > A-type K(+) current (IA). In addition, AIP (a selective CaMKII peptide inhibitor) and KN-93 caused a similar inhibition of INa-T and IK. Those evidences show that the excitability of capsaicin sensitive small and medium TG neurons can be regulated by Ca(2+)-CaM-CaMKII pathway through modulating VGSCs and VGPCs. Considering the specific distribution of CaMKII and its susceptibility to many analgesic stimuli, Ca(2+)-CaM-CaMKII pathway may play an important role in the peripheral sensory transduction, especially in nociception.

Diabetic neuropathy enhances voltage-activated Ca2+ channel activity and its control by M4 muscarinic receptors in primary sensory neurons.

Painful neuropathy is one of the most serious complications of diabetes and remains difficult to treat. The muscarinic acetylcholine receptor (mAChR) agonists have a profound analgesic effect on painful diabetic neuropathy. Here we determined changes in T-type and high voltage-activated Ca(2+) channels (HVACCs) and their regulation by mAChRs in dorsal root ganglion (DRG) neurons in a rat model of diabetic neuropathy. The HVACC currents in large neurons, T-type currents in medium and large neurons, the percentage of small DRG neurons with T-type currents, and the Cav3.2 mRNA level were significantly increased in diabetic rats compared with those in control rats. The mAChR agonist oxotremorine-M significantly inhibited HVACCs in a greater proportion of DRG neurons with and without T-type currents in diabetic than in control rats. In contrast, oxotremorine-M had no effect on HVACCs in small and large neurons with T-type currents and in most medium neurons with T-type currents from control rats. The M(2) and M(4) antagonist himbacine abolished the effect of oxotremorine-M on HVACCs in both groups. The selective M(4) antagonist muscarinic toxin-3 caused a greater attenuation of the effect of oxotremorine-M on HVACCs in small and medium DRG neurons in diabetic than in control rats. Additionally, the mRNA and protein levels of M(4), but not M(2), in the DRG were significantly greater in diabetic than in control rats. Our findings suggest that diabetic neuropathy potentiates the activity of T-type and HVACCs in primary sensory neurons. M(4) mAChRs are up-regulated in DRG neurons and probably account for increased muscarinic analgesic effects in diabetic neuropathic pain.

Nitric oxide inhibits nociceptive transmission by differentially regulating glutamate and glycine release to spinal dorsal horn neurons.

Nitric oxide (NO) is involved in many physiological functions, but its role in pain signaling remains uncertain. Surprisingly, little is known about how endogenous NO affects excitatory and inhibitory synaptic transmission at the spinal level. Here we determined how NO affects excitatory and inhibitory synaptic inputs to dorsal horn neurons using whole-cell recordings in rat spinal cord slices. The NO precursor L-arginine or the NO donor SNAP significantly increased the frequency of glycinergic spontaneous and miniature inhibitory postsynaptic currents (IPSCs) of lamina II neurons. However, neither L-arginine nor SNAP had any effect on GABAergic IPSCs. L-arginine and SNAP significantly reduced the amplitude of monosynaptic excitatory postsynaptic currents (EPSCs) evoked from the dorsal root with an increase in paired-pulse ratio. Inhibition of the soluble guanylyl cyclase abolished the effect of L-arginine on glycinergic IPSCs but not on evoked monosynaptic EPSCs. Also, inhibition of protein kinase G blocked the increase in glycinergic sIPSCs by the cGMP analog 8-bromo-cGMP. The inhibitory effects of L-arginine on evoked EPSCs and high voltage-activated Ca(2+) channels expressed in HEK293 cells and dorsal root ganglion neurons were abolished by blocking the S-nitrosylation reaction with N-ethylmaleimide. Intrathecal injection of L-arginine and SNAP significantly increased mechanical nociceptive thresholds. Our findings suggest that spinal endogenous NO enhances inhibitory glycinergic input to dorsal horn neurons through sGC-cGMP-protein kinase G. Furthermore, NO reduces glutamate release from primary afferent terminals through S-nitrosylation of voltage-activated Ca(2+) channels. Both of these actions probably contribute to inhibition of nociceptive transmission by NO at the spinal level.

Some subtypes of endocannabinoid/endovanilloid receptors mediate docosahexaenoic acid-induced enhanced spatial memory in rats.

ω-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) enhances cognitive functions; however, the underlying molecular mechanism remains unclear. Compelling evidence suggests that the endocannabinoid/endovanilloid systems play a pivotal role in regulating cognitive function. Thus, to correlate the effect of DHA on cognitive performance with the expression of endocannabinoid and endovanilloid receptors, we supplemented the diet of rats with DHA and performed in vitro experiments that focused on the endocannabinoid/endovanilloid receptors. We found that in vivo supplementation with an appropriate dose of DHA (150 or 300mg/kg/d) significantly improved learning and memory but that a higher intake (600mg/kg/d) increased the risk of memory impairment. In addition, we found that some subtypes of endocannabinoid/endovanilloid receptors (cannabinoid [CB] and transient receptor potential vanilloid [TRPV] receptors) were regulated in vitro by different concentrations of DHA in primary hippocampal neuron culture medium. Real-time polymerase chain reaction and western blot analysis showed that expression of both CB1 and TRPV1 was upregulated in a dose-dependent manner and reached a maximum level at 30µmol/L (CB1) and 60µmol/L (TRPV1) DHA. However, TRPV2 expression was downregulated in a dose-dependent fashion, and the peak of TRPV2 suppression was observed at 60µmol/L. The dose-dependent effects of DHA on the expression of these receptors were well correlated with DHA's effect on spatial memory. Meanwhile, CB2, TRPV3, and TRPV4 expressions were not altered at diverse concentrations of DHA. We concluded that some subtypes of endocannabinoid/endovanilloid receptors might be involved in enhanced spatial memory induced by DHA supplementation.