Herewith, we have analysed eQTL segregation in an outbred Duroc population using two groups of animals with divergent fatness profiles. This approach is particularly suitable to analyse the within-breed component of eQTL variation, with a special emphasis on loci involved in lipid metabolism.The main goal of the present study was to analyse the genetic architecture of mRNA expression in muscle, a tissue with an outmost economic importance for pig breeders. Previous studies have used FGeneChip Porcine Genome arrays (Affymetrix) were used to determine the mRNA expression levels of gluteus medius samples from 105 Duroc barrows. A whole-genome eQTL scan was carried out with a panel of 116 microsatellites. Results allowed us to detect 613 genome-wide significant eQTL unevenly distributed across the pig genome. A clear predominance of trans- over cis-eQTL, was observed. Moreover, 11 trans-regulatory hotspots affecting the expression levels of four to 16 genes were identified. A Gene Ontology study showed that regulatory polymorphisms affected the expression of muscle development and lipid metabolism genes. A number of positional concordances between eQTL and lipid trait QTL were also found, whereas limited evidence of a linear relationship between muscle fat deposition and mRNA levels of eQTL regulated genes was obtained.trans and influences biological processes related with muscle development, lipid deposition and energy balance. The identification of the underlying causal mutations and the ascertainment of their effects on phenotypes would allow gaining a fundamental perspective about how complex traits are built at the molecular level.Our data provide substantial evidence that there is a remarkable amount of within-breed genetic variation affecting muscle mRNA expression. Most of this variation acts in  Genetical genomics studies performed in human have indicated that: (e.g. Duroc x Pietrain and Large White x Landrace). This experimental design is particularly suited to capture the between-breed component of genetic variation. Although useful, eQTL maps obtained in crossbreds do not provide an accurate account of the regulatory DNA variation segregating in purebred swine populations, where intensive selection, inbreeding, founder effects and genetic drift have strongly modified genetic diversity.So far, the few eQTL studies performed in pigs In the current work, we have analysed the segregation of muscle eQTL in two groups of Duroc pigs with divergent phenotypes for fat deposition traits. Important differences in the expression of cell differentiation, energy balance and fat metabolism genes between these groups had been detected in a previous study transcripts, a total of 613 genome-wide significant eQTL  affecting the mRNA expression of 569 probes were identified (Sus Scrofa Chromosomes (SSC) 3 and 5 harbouring the highest number of eQTL (74 and 107 respectively). In contrast, only three and seven eQTL were observed on SSC11 and SSC14, respectively. Out of the 569 total eQTL-targeted probes, 490 were successfully mapped to the pig genome  were retained for further analysis  pigs, but no studies on the segregation of eQTL in porcine purebred population have been carried out so far. As a whole, the number and magnitude of muscle eQTL detected in the present study were comparable to those reported by Steibel et al. P-value cut off (P<0.0001), which is slightly more conservative than ours (P<0.0009). Similarly, Liaubet et al. i.e. many small-effect alleles are missed because of a lack of statistical power to detect them). This is particularly meaningful in a purebred population such as the one described in the current work, where allelic frequencies could have been substantially modified by selection, inbreeding and genetic drift.Strong evidences of muscle eQTL segregation had been previously reported by Steibel et al. 2 crosses, it is relevant to highlight that in previous studies we detected moderate to high heritability estimates 2 divergent crosses Although genetic variability of our Duroc population should be much lower than that detected in Ftrans-acting than cis-acting eQTL (396 vs 59 respectively) in the pig muscle. Following Ponsuksili et al. cis-acting when both the eQTL peak and the target gene fell within the interval delimited by the two flanking markers of the eQTL , so they would be more difficult to detect. Moreover, multiple-testing thresholds are stricter for trans-eQTL than for cis-eQTL cis-acting) eQTL were in general much more significant  than trans-acting eQTL. In summary, given the conservative thresholds applied to correct for multiple testing and the smaller effects expected for the trans-eQTL, a high number of trans-regulatory polymorphisms might be undetected in studies burdened by low sample size and/or less powerful designs. However, and in strong contrast with this theoretical expectation, our study and others trans- vs cis-eQTL. Importantly, this predominance of trans-eQTL decreases substantially when more stringent P-value cutoffs are employed to assess the significance of the data. For instance, when Steibel et al. P-value of 3.5×10−6 (corresponding to a FDR of 10%), 40 out of 62 significant eQTL had linkage peaks on the same chromosome where the regulated probe was physically located. Results obtained in the current study were also consistent with this statement: the predominance of trans- over cis-eQTL decreased dramatically as the significance threshold increased . Another plausible hypothesis that would be worth to investigate is whether a relatively lower genetic variation, as expected in our outbred population subjected to selection, could result in gene regulatory networks (trans-eQTL) standing out when compared to gene regulatory elements (cis-eQTL). In any case, the higher frequency of trans-eQTL probably reflects a biological reality since, as discussed above, experimental factors usually lead to underestimate the number of trans-eQTL. In this sense, Cheung et al. trans-eQTL are also more abundant than the cis-ones in human.The relative importance of ncreased , althougtrans-eQTL are expected to affect the expression of many genes while cis-eQTL must (necessarily) have a more restricted effect. A detailed analysis of eQTL affecting the expression level of several genes led us to define 11 eQTL hotspots .Studies in other organisms have identified hotspots containing genetic variants that influence multiple gene expression phenotypes regulation of fatty acid oxidation, fatty acid biosynthetic process, regulation of fatty acid metabolic process, regulation of lipid metabolic process, and lipid biosynthetic process among GO terms significantly enriched by our list of eQTL-regulated genes. Additionally, a variety of developmental and morphological processes related to muscle development and function  were also spread over the list of most significantly enriched GO terms. Other metabolic processes such as regulation of glucose metabolic process and regulation of insulin-like growth factor receptor signalling appeared in the middle of the list. Concerning the molecular function GO category  only indicated that most genes encoded intracellular components. Our findings partially agree with those obtained by Liaubet et al. GO term annotations were used in order to perform a functional categorization of genes regulated by genome-wide significant eQTL . As a prcategory , carboxycis-  and 16 trans-regulated  loci. In general, cis-regulated genes were involved in either (i) modulation of intracellular levels of bioactive lipids that function as second messengers to cell and differentiation events, or (ii) lipid transport by lipoproteins. The list of 16 trans-regulated loci included well-studied genes related with fat deposition  and meat tenderisation (CAST).Within the general goal of investigating the segregation of muscle eQTL at the within-breed level, we were particularly interested on the genetic regulation of lipid metabolism and fatness traits. On the basis of results previously presented, eQTL-regulated genes potentially associated with muscle fat deposition and meat quality traits were further investigated. First of all, we performed a more refined analysis of the genes enriching the five lipid metabolism-related GO terms  and 4. Thypertrophic cardiomyopathy and the adipocytokine signalling pathway on the top of the list. The insulin signalling pathway, associated with energy balance, was enriched by genes related to insulin resistance (e.g. IRS1) as well as by several genes involved in lipogenesis and cholesterogenesis . Besides, the PPAR signalling pathway appeared in the middle of the list, gathering several genes with roles in lipid biosynthesis and fatty acid uptake, transport, and metabolism . Finally, the TGF-beta signalling pathway, which regulates the switch of myocyte/adipocyte differentiation, was also present in this list.As a complementary approach, we performed a pathway analysis of the eQTL regulated genes. In clear consistency with the GO analysis, pathways involved in lipid metabolism and/or adipose/muscle tissue differentiation were amongst the most significant ones . These iTaken as a whole, these findings suggest that genetic factors modulating muscle lipid metabolism exert their effects at multiple levels including lipid biosynthesis, absorption, transport and degradation. However, the specific polymorphisms involved in the genetic control of these processes may vary from population to population. In their experiment, closely paralleling ours, Steibel et al. cis- and 267 trans-eQTL) out of 455 eQTL-regulated loci matched to genes that happened to be differentially expressed between pigs displaying divergent fatness profiles e.g. ACACA, ACSL1, ACSL5, ACLY, FABP5, CEBPD, IGFBP5 and SCD) that showed at least one trans-regulatory polymorphism. This result suggests that the divergent fatness phenotypes characterized in our purebred Duroc population by Canovas et al. i.e. they might be due to the differential segregation (in the High and Low groups) of regulatory polymorphisms with effects on lipid metabolism-related genes. Further research dealing with differential allelic segregation analyses at a whole-genome level would shed light on this topic.Finally, it is worth mentioning that 306  and a dataset of QTL for IMF, muscle cholesterol content and fatty acid profile segregating in the same Duroc population en genes  includincis-and trans-acting eQTL  accumulation, having been linked to susceptibility to the metabolic syndrome in humans QTL affecting muscle fatty acid composition  at SSC3, SSC6, SSC8, SSC14 and SSC18 also co-localised with several ing eQTL  and 4. Ats ARNTL . ARNTL ii.e. IMF content and composition traits), probably through the regulation of a number of genes related with lipid metabolism and other biological functions  out of 80 genes that were regulated by eQTL mapping to the confidence interval of QTL were selected for the correlation study, on the basis of their functional relationship with muscle fat deposition. We computed the correlation coefficients between their mRNA expression levels and muscle fat deposition traits, including IMF, muscle cholesterol content and fatty acid composition. Correlation coefficients that were significant in at least one of the three calculations (whole dataset or within High or Low fatness groups) are shown in NEDD4L mRNA levels and myristic fatty acid content, and between CYP24A1 expression levels and palmitic fatty acid content. However, consistency between correlation coefficients obtained within the two groups of pigs with divergent profiles for lipid traits was, in general, quite low. For instance, the mRNA levels of the ACACA and ACLY genes showed positive correlation coefficients with IMF content, but this relationship did not reach statistical significance in the High group. Similarly, muscle gene expression of IL12RB2 was positively correlated with IMF content but only in the High group . Opposite correlation relationship in extreme groups had been reported before between SCD protein expression levels and oleic acid muscle composition A correlation study was performed in order to confirm the relationship between mRNA expression of certain genes and phenotypic variation. A total of 16 (gh group . Even moi.e. limited sample size) and biological  factors. In this sense, it is well known that obesity and leanness, as “metabolic states”, can influence the expression of many genes, including those that are completely unrelated with lipid metabolism Taken as a whole, this correlation study provided limited evidences of a linear relationship between muscle fat deposition and mRNA levels of genes functionally related with lipid metabolism. Discrepancies between correlations estimated in the two groups might be explained by experimental .An experimental pig population  distributed in five half-sib families and four contemporary batches was generated from a commercial Duroc line mainly devoted to the production of fine quality dry-cured products. This commercial line was selected to increase intramuscular fat (IMF) content, jointly with prolificacy and growth traits, while maintaining carcass fatness. Inbreeding was controlled by an appropriate mating design and the sporadic introgression of genetic material. Pigs were castrated and controlled at IRTA-Pig Control Centre facilities. A number of phenotypes related with fatness, serum lipid levels, and IMF content and composition were recorded and measured as previously described by Gallardo et al. The Duroc commercial population was genotyped for 116 informative microsatellites covering the 18 autosomes with an average interval space between markers of 19.2 cM. All the information related to microsatellite genotyping and the description of the linkage map can be found in Quintanilla et al. gluteus medius muscle samples obtained from 105 Duroc pigs distributed in two groups with divergent phenotypes for fatness traits . Groups were established on the basis of multivariant analyses for several lipid deposition traits, as described in Cánovas et al. GeneChip Porcine Genome® arrays  procedures are also reported in Cánovas et al. http://linus.nci.nih.gov/BRB-ArrayTools.html. Microarray data obtained in the current study have been deposited in the Gene Expression Omnibus (GEO) public repository with GSE19275 and GSE26091 GEO accession numbers.We have analysed the global mRNA expression profile of Before performing the eQTL scan, the “minimum fold-change” filter implemented in the BRB-ArrayTools was applied in order to filter out those probes displaying low expression variability across the 105 muscle samples. The criterion consisted on selecting those probes with more than 20% of its expression values (from the 105 arrays) meeting at least ±1.5 times the median expression of the probe.Affymetrix probes which had passed the “minimum fold-change” filter described above. These eQTL analyses were performed for each probe, using the aforementioned panel of 116 microsatellites, by means of the GridQTL software http://www.gridqtl.org.uk/. The common model used for eQTL scanning was:ijky is the expression data of individual k; ib is the effect of thi contemporary batch of fattening (four levels); jl is the laboratory j effect (two levels); α is the regression coefficient ; kp is the probability of individual k inheriting a given allele from its common parent; ijke is the residual effect. Genome-wide significance thresholds for the F-values (eQTL model vs no eQTL model) were approximated with the Bonferroni correction as described in Gallardo et al. P-values of 0.0009 and 0.0002 respectively.A genome-wide eQTL scan was carried out for 6,139 Affymetrix porcine probe-sets (11 probes per probe-set). These probes were assembled to the pig genome sequence with the Ensembl annotation tool (http://www.ensembl.org/info/data/ftp/index.html) using CLCBio workbench software , and quality of all significant probes was thoroughly examined. In the mapping analysis, we took into account the proportion of mismatches with the reference sequence. In this way, probes were classified into uniquely mapped probes and non-specifically mapped probes.For those target probes regulated by significant eQTL, we analysed the http://www.animalgenome.org/cgi-bin/gbrowse/pig/), was used to locate all probes affected by significant eQTL as well as to align these eQTL to the pig genome. The alignment of eQTL genomic locations against target transcripts or genes was accomplished by converting the linkage eQTL map (cM) to genome sequence assembly locations (bp) as described in Hu et al. vs bp ratio, and the offset of the eQTL location to that of anchoring marker cis-acting eQTL (close to the target gene) and trans-acting eQTL (in different chromosome or far away from the target gene). As in Ponsuksili et al. cis-acting eQTL were defined as those in which the target gene position falls within the interval delimited by the two flanking markers of the eQTL peak.The GBrowse tool, available at the pig QTL database .Gene Ontology (GO) describes the basic hierarchies and relationships between terms within the context of biology. The three GO categories  were analysed in order to achieve a full coverage of the GO spectrum. The GO term enrichment analyses for overrepresentation of specific GO terms were carried out with the Database for Annotation, Visualization and Integrated Discovery (DAVID) bioinformatic package, available at Correlation analyses between expression levels and several phenotypes related to lipid metabolism and fat deposition were additionally carried out for a number of eQTL-regulated genes selected on the basis of the former analyses. The correlation study was performed using the CORR procedure of SAS  after adjusting phenotypes and expression levels for the environmental significant effects considered in previous analyses . Two correlations analyses were carried out: 1) correlation analyses considering the whole dataset (n = 105 for each correlation); and 2) within-group correlation analyses, considering separately the two groups of individuals with divergent lipid metabolism profiles .Table S1Genome-wide significant eQTL with high quality target probes: eQTL peaks, target gene locations, flanking markers, confidence intervals, distances from the closest marker, and classification as cis-/trans- eQTL.(XLS)Click here for additional data file.


Endoscopic ultrasound-guided fine needle aspiration-biopsy is the most widely used approach for cytological and histological material sampling in these situations used in up to two thirds of patients with pancreatic cancer. Based on this unique material, we and others developed strategies to improve the differential diagnosis between carcinoma and inflammatory pancreatic lesions by analysis of KRAS oncogene mutation, microRNA expression and methylation, as well as mRNA expression using both qRT-PCR and Low Density Array Taqman analysis. Indeed, differentiating pancreatic cancer from pseudotumoral chronic pancreatitis remains very difficult in current clinical practice, and endoscopic ultrasound-guided fine needle aspiration-biopsy analysis proved to be very helpful. In this review, we will compile the clinical and molecular advantages of using endoscopic ultrasound-guided fine needle aspiration-biopsy in managing pancreatic cancer.Endoscopic ultrasound-guided fine needle aspiration-biopsy is a safe and effective technique in diagnosing and staging of pancreatic ductal adenocarcinoma. However its predictive negative value does not exceed 50% to 60%. Unfortunately, the majority of pancreatic cancer patients have a metastatic and/or a locally advanced disease ( The five year survival rate after diagnosis is less than 3.5% ,2. Only 2.2.1.i.e., microbiopsy). A portion of this material, collected following air or saline flushing of the needle once the core biopsies have been reclaimed for histopathology, can be used for further molecular analysis. In our experience, a mean of 500 nanograms of DNA (range 150 nanograms to 1.5 micrograms) is obtained and DNA amplification is possible in all cases [Despite using fine needles of 22G, sufficient materials can be obtained for cytology and histology  and frozen at −20 °C until use. Despite all these precautions, difficulties persist. Quality and quantity of RNA must be systematically checked with bioanalyzers . Our group estimates that RNA samples that are highly degraded (RNA 18S/28S ratio less than 1) and/or in low quantities (lower than 100 picograms per μL) are usually not suitable for further analysis (mainly qPCR). However, even if quantity of RNA is low (but not degraded) RNA amplification kits are now available and permit up to 500 fold amplification with satisfactory reproducibility and reliability. In other terms, the RNA amplification from EUS-FNA material preserves the pattern of gene expression and is not influenced by the origin of the sample.3.KRAS mutation (75% to 95%) and to a lesser extent p16, DPC4 and p53 gene mutations [KRAS mutation was found in 60% to 65% of PDAC [p16 and DPC4 mutation analysis in pure pancreatic juice does not improve the sensitivity and specificity of KRAS mutation detection for the distinction of PDAC from CP [KRAS mutation could be detected in cellular materials obtained by EUS-FNAB [KRAS mutation analysis, EUS-FNAB appeared to be highly accurate for the differentiation of benign versus malignant pancreatic solid lesions [KRAS mutation analysis might be essential to strengthen the diagnosis of pancreatic masses by EUS. This latter point is essential for the differential diagnosis between PDAC and CP in the subgroup of pseudo-tumorous forms. Results of these studies, including our experience, are detailed in KRAS mutation assay alone are similar regardless of the technique used. In addition, DNA extraction has been performed on various types of samples such as cellular materials air blushed from the needle biopsy [KRAS mutation assay were also similar regardless of the method of sampling. Finally, the sensitivity and overall accuracy of PDAC diagnosis is improved when combining cytopathology and KRAS analysis ; the latter result reinforces the specificity of the ≪KRAS assay≫ for PDAC [KRAS analysis is now widely available due to its application as a predictive marker for anti-EGFR therapy in colon cancer, this diagnostic tool could also be applied to help the clinician in managing pancreatic masses. We are making extensive efforts to transfer this technique to clinical practice, as a rapid and cheap determination of KRAS mutation analysis using a Taqman-based allelic discrimination protocol.However, for PDAC ,28. Sinc4.PLAT and LCN2 found in resected tissues following macroarray analysis [modus operandi may require RNA amplification. Steg et al. used this approach to assay the mRNA levels of the Hedgehog pathway molecules in pancreatic carcinomas samples. They performed EUS-FNA biopsies before and during chemoradiation. While this work demonstrated the feasibility of this technique, no significant changes were observed in the expression of Hedgehog pathway molecules in response to chemoradiation [a la carte≫ treatment for epithelial cancers. Recently, it has been demonstrated that patients treated with gemcitabine-based adjuvant chemotherapy, the tumor expression levels of molecules that regulate gemcitabine intracellular metabolism  could predict the gemcitabine sensitivity (36). This strategy could also be applied in patients for whom gemcitabine-based palliative chemotherapy is proposed, by measuring the expression level of these molecules within the tissue material obtained by EUS-FNAB before treatment.Large scale analysis of gene expression has been widely proposed as a powerful method for malignancy diagnosis, predicting invasion and metastasis through the identification of biomarkers. Indeed, PDAC has previously been the focus of such studies, including our own experience -32. An ianalysis . Furtheranalysis . These radiation . In the et al. demonstrated that mutational status of a given gene is feasible in EUS-FNA samples; however they failed to associate the presence of somatic mutations of EGFR, or elevated copy numbers of EGR gene with the prognosis of PDAC [et al. reported that miRNA levels are affected in PDAC FNAs [let-7 expression is repressed in PDAC FNAs [Several teams succeeded in DNA analysis on EUS-FNA samples to investigate EGFR kinase mutations, EGFR gene amplification, as well as p16, p53 mutations of allelic losses at 19p (bearing p16 tumor suppressor gene) or 18q (bearing DPC4 tumor suppressor gene) chromosomes ,38. Thes of PDAC . In addi of PDAC ,38. Alon of PDAC . We and  of PDAC ,41. SafrDAC FNAs . The comDAC FNAs . The lev5.KRAS mutation analysis is highly promising and ready for use at a clinical level. Other molecular markers (including miRNAs) are yet to be characterized. Moreover, EUS-FNAB allows sampling of biological material from patients with locally advanced PDAC that, unfortunately, represent the vast majority of patients diagnosed with this cancer in clinical practice. In this way, cancer scientists have a unique access to tumor material to perform molecular investigations to better understand the physiopathology, the carcinogenesis, and the response to treatment of advanced PDAC.EUS-FNAB is a safe and effective technique for diagnosing PDAC, but its accuracy varies widely. However sampling cytological and histological materials is achieved in 85% of patients with PDAC and permits cytopathological diagnosis. The improvement of molecular biology including DNA and RNA amplification enables the analysis and the quantification of candidate molecular markers in cytological samples collected from EUS-FNAB. Analysis can be performed on cellular material sampled by air blushing or rinsing needle once core biopsy is reclaimed. In this way, the results of molecular analysis can be correlated to histology done on the tissue materials collected during the same needle pass. Thus, molecular analysis of EUS-FNAB material is a complementary tool for the diagnosis of PDAC (


Glutamate is the primary excitatory transmitter of sensory transmission and perception in the central nervous system. Painful or noxious stimuli from the periphery ‘teach’ humans and animals to avoid potentially dangerous objects or environments, whereas tissue injury itself causes unnecessary chronic pain that can even last for long periods of time. Conventional pain medicines often fail to control chronic pain. Recent neurobiological studies suggest that synaptic plasticity taking place in sensory pathways, from spinal dorsal horn to cortical areas, contributes to chronic pain. Injuries trigger long-term potentiation of synaptic transmission in the spinal cord dorsal horn and anterior cingulate cortex, and such persistent potentiation does not require continuous neuronal activity from the periphery. At the synaptic level, potentiation of excitatory transmission caused by injuries may be mediated by the enhancement of glutamate release from presynaptic terminals and potentiated postsynaptic responses of AMPA receptors. Preventing, ‘erasing’ or reducing such potentiation may serve as a new mechanism to inhibit chronic pain in patients in the future. While we work to survive in a competitive society, and seek different forms of pleasure, we try to avoid pain when it is possible. The study of pain is mainly driven by humans' desire to avoid or control pain. The discovery of analgesic drugs such as opioids has greatly helped doctors to reduce the suffering of patients during and after surgery. However, while these drugs are effective for controlling pain which is short lasting , it is ineffective in controlling chronic pain. In addition, opioids have many unwanted side effects after prolonged use. Recent integrative neuroscience studies have found that chronic pain and acute pain operate through different central mechanisms . InhibitLTP, the primary experimental model for studying the synaptic basis for learning and memory ,4, has b2.The study of pain benefits from the understanding of peripheral and spinal physiology. It has been known that selective sensory fibres and receptors are important for the initiation of pain transmission. Nerve activity caused by activation of nociceptors conveys to selective cortical regions that aid the formation of pain perception and unpleasantness of pain . Cumulat3.In vivo electrophysiological recordings of neurons from animals as well as humans found that ACC neurons respond to peripheral noxious stimuli, and show increased responses to greater intensity of pain. This key finding is further supported by numerous human imaging studies in both normal individuals and patients with chronic pain. Human brain imaging studies further demonstrate that ACC neurons can be ‘activated’ in emotional pain conditions such as sadness due to loss of loved ones or emotional divorce. Animal behavioural studies using pharmacological agents find that inhibition of ACC activity is analgesic in different animal models of acute pain as well as chronic pain. Although studies using direct manipulations in human brains are limited, some clinical reports using electronic lesions or surgical ablation can reduce chronic pain caused by cancer [Among several cortical areas , the ACCy cancer . Cogniti4.Hyperalgesia and allodynia are two major forms of sensitized behavioural responses measured in the animal models of chronic pain, such as inflammatory pain and neuropathic pain. In the case of hyperalgesia, nociceptive responses to noxious stimuli are significantly enhanced, and in allodynia, previously non-noxious stimuli cause behavioural nociceptive responses such as cold stimuli or non-noxious mechanical touch or brush . At the Differing from behavioural allodynia and hyperalgesia, spontaneous pain has been less frequently investigated. This is because of the lack of appropriate behavioural models. Recent studies showed that animal place avoidance test can be used to evaluate spontaneous pain or tonic pain ,12. It i5.N-methyl-d-aspartate) receptors is required for LTP [et al. [Studies suggest LTP in spinal dorsal horn neurons is believed to be the key mechanism for potentiation of sensory transmission after injury. Previous electrophysiological studies consistently indicated that spinal responses to sensory stimuli including non-noxious stimuli are enhanced. Electrical stimulation of sensory fibres or peripheral injury indeed induced LTP-like enhancement in spinal dorsal horn neurons . LTP hap for LTP . Using t [et al.  reportedIn addition, the activation of NMDA receptors and application of serotonin (5-HT) or 5-HT receptor agonists induced long-term facilitation of synaptic response ,16, a he6.in vivo recordings of freely moving mice during trace fear learning, indicating that the TBS protocol mimics physiological conditions in the ACC [It is commonly thought that mature cortical synapses are less plastic. In certain cortical areas, LTP is found to be age-dependent under experimental conditions . The dis the ACC . In addi the ACC .Table 17.in vitro brain slice conditions .Pharmacological studied using selective receptor antagonists reveal that ACC LTP exists in at least four different forms: NMDA receptor-dependent, L-type voltage-gated calcium channel (L-VGCC)-dependent, late-phase LTP (L-LTP) and presynaptic LTP (pre-LTP) under experimental nditions . Some of(a)NMDA-receptor-dependent LTP has been commonly reported in central synapses ,21. In A(b)L-VGCC-dependent LTP has been reported in the hippocampus . L-VGCCs(c)et al. 2014, unpublished data; see [Recent studies using a 64-channel multi-electrode array (MED64) show that ACC LTP induced by multiple TBS can last more than 5 h . This foata; see  for the ata; see . Future (d)To date, most forms of LTP reported in the ACC are postsynaptically induced. Our recent study reported that a new pairing protocol can induce LTP in the ACC that is purely presynaptically induced ,25. This8.(a)2+ serves as an important intracellular signal for triggering a series of biochemical events that contribute to the induction of ACC LTP. Postsynaptic injection of BAPTA into ACC neurons completely blocked the induction of LTP, indicating the importance of elevated postsynaptic Ca2+ concentrations [12 are critical for the induction of cingulate LTP [Catrations . Studieslate LTP .(b)Cyclic adenosine monophosphate (cAMP), produced by adenylyl cyclases (ACs), is a key second messenger in neural systems. Among more than 10 subunits, AC1 is the key AC subtype that responds positively to calcium–CaM . Within (c)2+ activates CREB involves the Ca2+/CaM-dependent protein kinase type IV (CaMKIV). CaMKIV is distinguished among the CaM kinases in its capacity to activate CREB-dependent transcription both by virtue of its nuclear localization and catalysis of CREB phosphorylation on serine 133. CaMKIV is enriched in the ACC [Activity-dependent activation of immediate early genes such as cAMP response element-binding protein (CREB) is critical for synaptic plasticity ,31. A ma the ACC , and gen the ACC . The req(d)The extracellular signal-regulated kinase (ERK) in the mitogen-activated protein kinase (MAPK) cascade has been implicated in synaptic plasticity and is known to be activated in ACC neurons after injury . In the 9.Under different physiological and pathological conditions, including fear conditioning, peripheral inflammation or nerve injury, several key immediate early genes and their related products are activated in the ACC neurons. They include CREB, Egr1 and fragile X mental retardation protein (FMRP). It is likely that they mainly contribute to L-LTP, although their possible roles in LTP of the first hour after induction cannot be completely ruled out.(a)In the ACC, CREB is activated in an activity-dependent manner, and is downstream from key intracellular messengers that are important for ACC LTP, such as AC1 and CaMKIV ,32. Rece(b)The zinc finger transcription factor Egr1  is critical for coupling extracellular signals to changes in cellular gene expression. The upstream promoter region of the Egr1 gene contains binding sites for cyclic AMP-response elements (CREs), suggesting that Egr1 may act downstream from the CREB pathway. In ACC neurons, Egr1 activity is triggered by injury including peripheral inflammation or digit amputation . One pos(c)FMRP is a ubiquitously expressed mRNA binding protein associated with polyribosomes and is thought to be involved in the translational efficiency and trafficking of certain mRNAs ,39. FMRP10.2+-permeable GluA2-lacking receptors contribute to the maintenance of LTP and are necessary for subsequent LTP stabilization. GluA2/3 subunits may continually replace synaptic GluA2/3 subunits in an activity-independent manner that maintains constant synaptic transmission. We also examined the role of these peptides in synaptic potentiation in the ACC and found that the GluA2/3–PDZ interaction had no effect on cingulate LTP. We did find that the same interfering peptides inhibited cingulate long-term depression (LTD) [A major form of LTP in the ACC requires activation of postsynaptic NMDA receptors. The expression of ACC LTP is likely mediated by postsynaptic modification/trafficking of AMPA receptors. The roles of GluA1  and GluA2/3  in ACC LTP have been investigated using genetic and pharmacological approaches. Postsynaptic injection of GluA1 subunit C-terminal peptide analogue Pep1-TGL blocked cingulate LTP , indicaton (LTD) . These fon (LTD) .11.Protein kinase M zeta (PKMζ) has been suggested as a key factor in maintaining LTP in the learning-related hippocampus ,46. In t12.in vitro study of LTP is its physiological or pathological relevance. One reason for studying ACC plasticity is its potential link to the injury-related central plasticity. Can LTP be detected in vivo in the ACC after peripheral injury? Indeed, peripheral digit amputation triggers significant and long-lasting enhancement of synaptic responses within the ACC [in vivo conditions, peripheral sensory activity is not important for potentiation, because a local anaesthetic, QX-314, injected into the hindpaw (5%) did not affect the synaptic potentiation in the ACC [In the brain slices, cingulate synapses can undergo LTP after the experimentally designed training protocols. One major question for  the ACC . Importa the ACC .in vivo, we performed intracellular recordings from anaesthetized rats. We found that there was a long-lasting membrane potential depolarization in ACC neurons of adult rats after digit amputation in vivo [Because it is difficult to determine the origin of electrical changes using field recordings  in vivo . Shortly in vivo .13.in vivo electrophysiology provides direct evidence for pathological relevance of LTP in the ACC, it is difficult for studying cellular and molecular mechanisms. Detecting synaptic changes in the brain slices of animals with chronic pain will provide us opportunities to study the basic mechanisms of chronic pain. In fact, both postsynaptic and presynaptic changes are reported in the brain slices of animals with inflammatory or neuropathic pain. Furthermore, in addition to changes in glutamate AMPA receptors, NMDA GluN2B receptors are also found to undergo LTP.Although (a)et al. [To detect possible changes in synaptic transmission within the ACC after nerve injury, AMPA-receptor-mediated EPSCs in pyramidal neurons in the layer II/III of the ACC were measured in mice with peripheral nerve ligation . The inpet al.  reportedet al. . These ret al. 2014, unpublished data).Biochemical and anatomic evidence further support the involvement of postsynaptic AMPA receptors . The mem(b)Previous studies using genetically manipulated NR2B (or GluN2B) mice found that chronic pain is significantly enhanced in transgenic mice –58. Does(c)In addition to postsynaptic enhancement, recent studies indicate that presynaptic release of transmitter is also enhanced in the ACC . Paired-In addition to the use of PPF in the ACC after peripheral nerve injury, AMPA-receptor-mediated miniature EPSCs (mEPSCs) in ACC neurons in the presence of 0.5 μM tetrodotoxin were also found to be affected. After peripheral nerve injury, there was an obvious increase of mEPSC frequency in ACC neurons compared with that of control groups. Considering most LTP studied in the ACC is postsynaptically expressed, it is important to develop an experimentally induced pre-LTP protocol to mimic presynaptic changes of glutamate release in the ACC after peripheral injury.14.Among several key second messengers that contribute to ACC LTP, AC1 is unique for LTP in this region. Previous studies using AC1 knockout mice as well as recent pharmacological studies of the AC1 inhibitor NB001 consistently showed that AC1 or AC1 activity is not essential for hippocampus LTP and behavioural learning ,55 figu. By contIn addition to producing analgesic effects by inhibiting the upregulation of AMPA receptors in chronic pain conditions, our recent studies found that AC1 activity is also critical for the upregulation of NMDA GluN2B receptors in the insular cortex . These f15.From a traditional point of view, there are many good reasons for scientists and drug developers to avoid studying the ACC for the treatment of chronic pain. The spinal dorsal horn and periphery are still seen as ‘gold’ targets for controlling pain. However, in the case of chronic pain, more and more evidence indicates that some central changes occur right after the injury. Just like fear memory and other learning process, it is too late to interfere at the periphery if such ‘bad’ memory takes place in the brain. Many failed surgical manipulations for chronic pain patients prove the case. Many e-mails from chronic pain patients I have received indicate that the current medical care systems, modern medicines as well as alternative medicine, often fail to help. These individuals include patients with spinal cord injury, cancer, neuropathic pain, etc. So far among drugs approved by the US Food and Drug Administration for the treatment of pain, there is no single class of pain medicine that targets injury-related plasticity.From a basic point of view, cumulative evidence demonstrates that pain or injuries activate important cortical areas such as the ACC, prefrontal cortex, insular cortex, amygdala and the hippocampus. The impact of such injury on emotion and cognitive functions is traditionally ignored. Here, I would like to propose four major possible research questions that I believe are highly relevant for ACC plasticity.in vivo, show that ACC–ACC synapses and thalamus–ACC synapses may both undergo LTP after training or injury [in vivo approaches, and/or in vitro preparations if possible, to investigate if LTP in these two different pathways onto cingulate neurons may share similar mechanisms.First, is LTP equally occurring in thalamic inputs and cortical–cortical connections ? Dual patch-clamp recordings show that cortical and cortical connections can even occur within local neurons located in different layers of cells within the ACC . Previour injury . It willSecond, is there any presynaptic form of ACC LTP? What are physiological or pathological functions of pre-LTP in the ACC? Electrophysiological and pharmacological studies reveal that there is presynaptic enhancement of glutamate releases in chronic inflammatory pain and neuropathic pain conditions. However, we also found that NMDA-receptor-dependent ACC LTP is purely postsynaptically expressed . Thus, iThird, what is the impact of synaptic LTP on neuronal coding within the ACC? One key question for synaptic plasticity is the impact of LTP on neuronal action potentials. Is LTP contributing to increases in neuronal firing to subsequent stimuli such as in the case of hyperalgesia and allodynia? Or does LTP contribute to special activity within a neuronal circuit?Finally, what is the relationship between pain-induced plasticity and emotional disorders in chronic pain conditions? Depression, anxiety, loss of hope may be triggered by injury at the synaptic level. These ‘side effects’ become amplified if pain sensation persists owing to the failure of treatment or medicine. Is there any role of ACC LTP in pain-related emotion?In summary, integrative approaches have yielded significant progress in our understanding of synaptic mechanisms for sensory transmission, modulation and plasticity in the ACC. The involvement of each signalling protein will be identified. The combination of different experimental approaches such as synaptic neurobiology, systems biology and neuronal circuits is clearly required to understand how synaptic plasticity affects human and animal behaviours under physiological and pathological conditions.


The lateral habenula (LHb) plays an important role in learning driven by negative outcomes. Many drugs of abuse, including ethanol, have dose-dependent aversive effects that act to limit intake of the drug. However, the role of the LHb in regulating ethanol intake is unknown. In the present study, we compared voluntary ethanol consumption and self-administration, yohimbine-induced reinstatement of ethanol seeking, and ethanol-induced conditioned taste aversion in rats with sham or LHb lesions. In rats given home cage access to 20% ethanol in an intermittent access two bottle choice paradigm, lesioned animals escalated their voluntary ethanol consumption more rapidly than sham-lesioned control animals and maintained higher stable rates of voluntary ethanol intake. Similarly, lesioned animals exhibited higher rates of responding for ethanol in operant self-administration sessions. In addition, LHb lesion blocked yohimbine-induced reinstatement of ethanol seeking after extinction. Finally, LHb lesion significantly attenuated an ethanol-induced conditioned taste aversion. Our results demonstrate an important role for the LHb in multiple facets of ethanol-directed behavior, and further suggest that the LHb may contribute to ethanol-directed behaviors by mediating learning driven by the aversive effects of the drug. The lateral habenula (LHb) has been importantly implicated in learning driven by adverse outcomes. Neurons in the primate LHb are excited by negative stimuli, such as an aversive air puff or cues predicting the absence of reward The positively reinforcing effects of drugs of abuse motivate further drug seeking, particularly in initial stages of drug use The neural circuitry through which the aversive effects of ethanol suppress voluntary consumption is not well defined. Evidence supporting a role for the LHb in learning driven by aversive outcomes, including those caused by other drugs of abuse, raises the possibility that this brain region could contribute to these suppressive effects. To begin characterizing the role of the LHb in ethanol-directed behaviors, we studied voluntary ethanol consumption, ethanol self-administration, yohimbine-induced reinstatement of ethanol seeking, and ethanol-induced CTA in LHb and sham-lesioned rats. Our results show that lesions of the LHb increase both voluntary ethanol intake and self-administration, block yohimbine-induced reinstatement of ethanol seeking, and attenuate a taste aversion conditioned by a single noncontingent ethanol injection.All procedures used were approved by the University of Utah Animal Care and Use Committee and carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.Ad libitum access to food and water was available throughout all experimental procedures. A summary of the experimental groups and the timeline of experimental procedures (described below) within each group is provided in 136 male Long-Evans rats  were used in the present experiments. Rats were single-housed in Plexiglas tub cages and maintained on a 12 hour light/dark cycle. Ethanol  solutions were prepared in filtered tap water to a concentration of 20% (v/v) for use in the intermittent ethanol access paradigm, and prepared in physiological saline to a concentration of 20% for the CTA experiment. Saccharin, quinine and yohimbine  solutions were prepared in distilled water.Surgical anesthesia was induced and maintained with isoflurane . The skull was exposed and burr holes drilled bilaterally above the LHb. Lesions were produced by passing current  through a stainless steel electrode  at two sites within each hemisphere that targeted anterior and posterior portions of the LHb. Coordinates for these sites (in mm from bregma) were: –3 and –3.7 posterior; 0.7 lateral; and –5.4 ventral. In sham-lesioned animals, electrodes were lowered to stereotaxic coordinates 1 mm dorsal to the LHb but no current was passed at the target site. Rats were allowed to recover for at least one week after surgery before experiments commenced.ad lib water was available. IEA was provided for a minimum of 8 weeks (24 drinking sessions). Ethanol and water intake were measured by weighing bottles before and after each 24 hour access period. These measures were used to calculate ethanol intake  and preference . Body weight and food intake were measured weekly during IEA.Voluntary ethanol consumption was measured using an intermittent ethanol access (IEA) two bottle choice paradigm In a subset of sham and lesioned rats  and aversion to quinine solutions  were assessed using a two-bottle choice paradigm in the home cage in a subset of sham and lesioned rats (n = 7 rats in each group). In this paradigm, a single bottle of tastant was made available for 48 hours in the home cage, concurrent with a single bottle of water; intake of the tastant and water were measured every 24 hours, and bottle positions were switched after the first 24 period. Intake during the two 24 hour periods was averaged to produce a single measure of consumption for each tastant concentration. Tastants were presented in sequential order of increasing concentration in consecutive 48 hour intervals. Intake during access to the quinine concentration series was measured first, followed by intake during presentation of the saccharin concentration series. To investigate the timeline of saccharin consumption over 24 hour access periods, intake of highly preferred 5 mM saccharin was measured during the four intervals in which ethanol intake was measured: 9–10 am, 10 am–6 pm, 6–7 pm, and 7 pm–9 am.After a minimum of two months home cage access to 20% ethanol in the IEA paradigm, rats (n = 10 sham and 9 lesioned rats) were trained to self-administer 20% ethanol. Operant chambers  were equipped with a central reward receptacle flanked by retractable levers, with illuminated cue lights over each lever. Operant chambers were enclosed in sound attenuating chambers equipped with fans that increased ventilation and provided masking noise. Ethanol was delivered to the reward receptacle via a programmable syringe pump.In an initial overnight session, rats were trained on an FR1 schedule to respond for 20% ethanol. In this overnight session, only the active lever was presented to facilitate learning. Each lever press caused the active lever to retract, the associated cue light to extinguish, and resulted in immediate delivery of 0.1 mL 20% ethanol into the reward receptacle. After 5 seconds, the active lever was extended and the cue light illuminated. After the initial overnight session, rats were trained in 1 hour sessions three times per week  using an FR1 schedule in which only the active lever was available. After 2 weeks, rats progressed to an FR3 schedule in which the session length was decreased to 30 minutes and the inactive lever was introduced. Lever presses on the inactive lever were recorded but had no programmed consequences. Responses in this paradigm were measured over two weeks before extinction training began (below).During extinction sessions, the syringe containing 20% ethanol was removed from the syringe pump, and thus active lever presses no longer resulted in ethanol delivery. In all other respects, the extinction paradigm was identical to the final operant response paradigm described above, including presentation of visual and auditory cues . Extinction sessions were run on alternate weekdays for four successive sessions before testing for yohimbine-induced reinstatement.Reinstatement was studied by administration of the α2 receptor antagonist yohimbine  or vehicle solution (distilled water) 30 minutes prior to testing in extinction sessions. Because yohimbine reliably induces multiple reinstatements of ethanol seeking Fifteen ethanol-naive rats (7 sham and 8 lesioned rats) were trained to operantly self-administer 2% sucrose. With the exception of the use of sucrose as a reinforcer, the paradigm used was identical to the final operant paradigm used in ethanol self-administration (30 minute FR3 task with both active and inactive levers available). Training in this group was similar to that described for ethanol self-administration above, and began with an initial overnight training session . In five subsequent sessions, the response requirement was maintained at FR1 but the session duration was shortened to a single hour. Finally, rats in this group progressed to the final paradigm  for ten successive sessions.Thereafter, extinction followed by reinstatement sessions were carried out. The extinction paradigm was identical to that described for extinction of ethanol seeking. A total of seven successive extinction sessions were run in this experiment. This increase (over four sessions used in extinction of ethanol seeking) was incorporated because response rates for sucrose self-administration were substantially higher than those occurring during ethanol self-administration. Yohimbine-induced reinstatement was tested in this experiment in a manner identical to that described above in studies of ethanol reinstatement.ad lib access to both food and water. We avoided water deprivation often used in CTA paradigms because dehydration causes anorexia A total of 79 ethanol-naïve animals  were included in the CTA experiment. After lesion surgery, all rats were single housed and allowed at least one week recovery in home cages. Thereafter, rats were habituated to handling for two days before beginning the CTA experiment. Throughout the experiment, rats were maintained with The first day of the CTA experiment, rats were given 30 minutes home cage access to supersaccharin. Rats were then assigned to saline or ethanol injection groups, with mean supersaccharin consumption matched between groups. Rats in the resulting four groups  were immediately injected with either ethanol  or saline (volume matched to ethanol injections) and returned to their home cages. The ethanol dose used was based on pilot studies showing it produced reliable but not saturating CTA. On each of the next six days, rats again received a single period of 30 minutes home cage access to the supersaccharin solution and consumption was measured. Supersaccharin access sessions, including the initial session, occurred between 4 and 6 pm daily.Blood ethanol concentration (BEC) was measured after voluntary ethanol intake in the IEA paradigm and operant self-administration sessions. BECs after ethanol consumption in the IEA paradigm were measured from tail vein blood collected after the first 30 minutes of ethanol access, the interval during which previous results To determine if LHb lesion altered ethanol metabolism, we measured BECs after noncontingent injection of ethanol  in a separate group of ethanol-naïve rats (4 sham and 4 lesioned).Blood samples were collected into heparinized capillary tubes at 15, 30, 60, 120 and 180 minutes after injection. For all BEC measurements, blood plasma was isolated from samples by perchloric acid precipitation and brief centrifugation . BEC was measured using the NAD-NADH enzyme spectrophotometric method Rats were deeply anesthetized with pentobarbital and perfused with physiological saline followed by 4% formaldehyde. Brains were cryoprotected and sectioned in 50 µm slices. Sections were mounted and Nissl stained. Damage to the LHb was localized by comparison to a reference atlas Though damage was largely confined to the LHb, some lesion sites encroached upon surrounding structures, including the medial habenula (MHb). To determine if damage to this structure contributed to voluntary ethanol intake in the IEA paradigm, we quantified the extent of this damage in each lesioned animal. The damage to the MHb in each hemisphere was estimated by visual inspection, and was scored as 0% (no damage), 25%, 50%, 75%, or 100% (complete ablation). Scores for each hemisphere were averaged to produce a single estimate of damage to the MHb within each rat. Rats were divided into high and low damage groups by performing a median split of this data, and ethanol intake in the IEA paradigm was then compared between groups.Voluntary ethanol intake, escalation of ethanol intake, taste preference, and reinstatement of ethanol and sucrose seeking were analyzed using two-way repeated measures ANOVA. All analyses included lesion (sham or LHb) as one factor. The second factor consisted of IEA drinking session (voluntary ethanol intake); time interval ; tastant concentration (taste preference); or drug . Extinction of ethanol-self administration and ethanol-induced CTA were analyzed using two- (factors of lesion and time) and three-way  ANCOVA, respectively. Baseline ethanol self-administration and supersaccharin intake were used as covariates in these analyses, respectively. Where appropriate, Holm-Sidak posthoc tests were used. BECs and operant self-administration were analyzed using Pearson’s correlation test and t-tests.Intermittent home cage access to 20% ethanol (IEA) in a two bottle choice paradigm resulted in steady escalation of ethanol consumption in both sham and lesioned animals , which ath drinking session and were intermittently significant thereafter . Total fluid intake (water plus ethanol consumption) did not differ between the two groups . Lesioned rats consumed more ethanol during this period  and reached significantly higher BECs (To determine if differences in voluntary ethanol consumption resulted in different blood ethanol concentrations (BECs), we measured ethanol concentration in tail vein blood collected after the first 30 minutes of ethanol access in the IEA paradigm. BEC measures were significantly correlated with ethanol consumption occurring during this period for both sham and lesioned rats  in a separate group of previously ethanol-naïve rats (n = 4 sham and 4 lesioned). BECs in these groups did not differ at any time point after ethanol injection  were allowed an initial 10 weeks of IEA, followed by nearly 7 weeks of abstinence (46 days), and then two additional weeks of IEA. Rats were maintained in their home cages during the period of abstinence with Gustatory function contributes to voluntary ethanol intake Saccharin preference increased as a function of increasing concentration . Though lesioned rats consumed more saccharin than sham rats in every time period except the first hour of access, this difference did not reach statistical significance   = 3.2, NS; and no significant interaction of lesion x time, F  = 1.2, NS). By contrast, lesioned rats consumed significantly more ethanol specifically during the first hour of ethanol access . Interestingly, these and previous results To further investigate elevated saccharin and ethanol intake in lesioned animals, we studied the timing of 5 mM saccharin and 20% ethanol ingestion during 24 hour periods of home cage access. The distribution of saccharin intake across the 24 hour cycle was similar for sham and lesioned rats, with both groups consuming saccharin at highest rates during the first hour of the dark cycle  with a history of IEA were trained to self-administer 20% ethanol on an FR3 schedule. Stable levels of responding (average of last three rewarded sessions) were significantly higher in lesioned vs. sham rats  or vehicle (distilled water) 30 minutes before testing in the extinction paradigm. Vehicle administration resulted in rates of operant responding very similar to those recorded in the final extinction session  after an initial 30 minute period of supersaccharin access in the home cage. Ethanol administration conditioned an aversion to supersaccharin in both sham and lesioned groups as demonstrated by reduced supersaccharin intake after ethanol injection , day 1, Lesions were largely confined to the LHb , though Our results show that the LHb plays an important role in controlling ethanol-directed behaviors. LHb lesion increased the rate at which rats escalated voluntary ethanol consumption in a two bottle choice paradigm, resulted in higher levels of maintained intake relative to sham rats both before and after a period of abstinence, and gave rise to higher BECs relative to sham animals. Operant self-administration of ethanol was also increased in lesioned rats, again resulting in higher BECs than those measured in sham animals. In addition, yohimbine-induced reinstatement of ethanol and sucrose seeking were blocked in animals with LHb lesions. Finally, our results show that ethanol CTA was significantly attenuated by LHb lesion. These results demonstrate an important role for the LHb in regulating ethanol-seeking and consumption and moreover show that LHb circuits contribute to learning driven by ethanol’s aversive effects.th week of IEA, at which time ethanol intake in lesioned rats was approximately 50% higher than that in the sham group. This difference in intake was maintained for the remainder of the IEA paradigm.LHb lesions did not acutely increase voluntary ethanol consumption, but rather increased the rate at which escalation of intake occurred, leading to higher sustained levels of ethanol consumption. Ethanol intake in the first week of the IEA paradigm was similar in sham and lesioned groups  and 2A. Changes in taste preference are unlikely to contribute to increased ethanol intake and self-administration in LHb-lesioned rats. Though lesions of the habenular complex have been reported to decrease quinine intake Our findings of increased ethanol intake and self-administration in LHb-lesioned animals contribute to a growing body of evidence implicating the habenula in regulating drug seeking behaviors. Habenular circuits have been shown to play a central role in regulating learning driven by negative outcomes Increased ethanol intake and self-administration in LHb-lesioned animals may also arise through deficits in learning from aversive drug outcomes. Ethanol causes dose-dependent aversive effects that include nausea, sedation, motoric impairment and hangover effects Given evidence of an important role for the LHb in aversive conditioning, lesion of this structure might be expected to impair extinction learning, particularly as lesion and stimulation of the LHb respectively impair and accelerate extinction of cocaine-seeking The effects of LHb lesion were not specific to reinstatement of ethanol seeking, as yohimbine-induced reinstatement of sucrose seeking was also blocked in lesioned animals. Our results extend recent findings that pharmacological inactivation of the LHb blocks yohimbine-induced potentiation of cue-dependent reinstatement of cocaine-seeking, as well as attenuating yohimbine-induced anxiety-related behaviors In summary, our study provides novel evidence of an important role for the LHb in a number of ethanol directed behaviors. Our results show that LHb lesions increase voluntary ethanol intake and operant-self administration and block yohimbine-induced reinstatement of ethanol seeking. Further, LHb lesions attenuate ethanol CTA. These results are likely to have important implications for mechanisms underlying alcohol use disorders as studies in human volunteers show that vulnerability to developing an alcohol use disorder is inversely related to sensitivity to acute ethanol effects, including the aversive effects of the drug


Rituximab has improved the treatment of B-cell non-Hodgkin lymphomas. While it is generally well tolerated, serious adverse effects including infusion reactions with hemodynamic compromise and cardiac arrhythmias or ischemia are possible. We report a case of coronary vasospasm occurring during a rituximab infusion in a patient with minimal tumour burden and who had no cardiac risk factors. This case highlights that determination of the cause of ischemia is important and may identify some patients who can be successfully rechallenged. Rituximab is a chimeric murine-human monoclonal antibody that binds specifically with CD20, which is expressed on the majority of mature B-cell non-Hodgkin lymphomas. In the last decade, use of this drug alone or in combination with chemotherapy has improved the outcomes of patients with these diseases. The most common adverse effect is immediate hypersensitivity reactions, but postmarketing reports have cited increased rates of rare but serious complications such as hepatitis reactivation, bowel perforation or obstruction, progressive multifocal encephalopathy, and cardiac arrhythmias or ischemia . We report on the case of documented coronary vasospasm during a rituximab infusion, occurring in the absence of other symptoms typical of an infusion reaction and in a patient with no coronary artery disease.Helicobacter pylori infection. A 52-year-old woman presented with epigastric pain, melena, and a five-kilogram weight loss. She has a past history of gastric ulcers and her father had died of gastric cancer. She had no anemia, but symptoms were persistent despite therapy with a proton pump inhibitor so she underwent a gastroscopy which revealed only a healing ulcer in the duodenum. Biopsy revealed follicular lymphoma (FL), grade 1-2. There was no dysplasia or  Staging computed tomography (CT) revealed only thickening of the second part of the duodenum with no significantly enlarged lymph nodes. Bone marrow biopsy was positive for involvement with FL. For symptomatic stage 4 disease, systemic treatment with rituximab, cyclophosphamide, vincristine, and prednisone (R-CVP) was recommended. Screening blood work revealed a history of hepatitis B infection with a positive core antibody so she was started on lamivudine for reactivation prophylaxis.Following administration of standard premedication with diphenhydramine 25 mg iv, acetaminophen 1000 mg po, and prednisone 100 mg po, the rituximab infusion was initiated prior to the other antineoplastic agents. Within 10 minutes of her first treatment, she developed retrosternal chest pain associated with dyspnea. She had no hypotension, fever, rigors, nausea, or skin changes. Vital signs were stable and unchanged from baseline. The infusion was held, nitroglycerin was given, and pain resolved after 15 minutes. She has no cardiac risk factors other than a seven pack-year smoking history, but ECG documented new onset T-wave inversion in the anterior precordial leads  and activation of the complement system [Four clinical trials have demonstrated that the addition of rituximab to chemotherapy results in improved survival in the first-line treatment of FL –5. Whilet system , 8. Pre-Cardiac events including hypotension, arrhythmias, and chest pain were observed in at least two previous phase II trials of rituximab alone but were attributed either to previously existing heart disease  or infusHowever, our study is the first to demonstrate an acute coronary syndrome (ACS) due to coronary vasospasm. Although chest pain resolved following discontinuation of the infusion other features typical of a hypersensitivity reaction were absent. Symptoms were responsive to nitroglycerin and were associated with reversible ECG changes and no cardiac enzyme elevation. She had minimal tumour burden, no significant cardiac risk factors, and normal coronary anatomy with no atherosclerotic disease.Coronary vasospasm has not been described during rituximab infusion but is a well-known side effect of continuous 5-fluorouracil infusion, which presents in a similar manner –15. The This case highlights coronary vasospasm as a previously unreported infusion-related adverse effect of rituximab. In patients who develop chest pain during rituximab administration, this possibility of ACS should be considered and appropriate cardiac investigation should be undertaken, even if patients have no history of or risk factors for cardiac disease. In the absence of significant coronary artery disease, rechallenging with rituximab may be possible following optimal premedication in a monitored setting in selected cases where benefit of rituximab overweighs the risk of recurrent of cardiac ischemia.


The ability to concentrate on relevant sounds in the acoustic environment is crucial for everyday function and communication. Converging lines of evidence suggests that transient functional changes in auditory-cortex neurons, “short-term plasticity”, might explain this fundamental function. Under conditions of strongly focused attention, enhanced processing of attended sounds can take place at very early latencies (~50 ms from sound onset) in primary auditory cortex and possibly even at earlier latencies in subcortical structures. More robust selective-attention short-term plasticity is manifested as modulation of responses peaking at ~100 ms from sound onset in functionally specialized nonprimary auditory-cortical areas by way of stimulus-specific reshaping of neuronal receptive fields that supports filtering of selectively attended sound features from task-irrelevant ones. Such effects have been shown to take effect in ~seconds following shifting of attentional focus. There are findings suggesting that the reshaping of neuronal receptive fields is even stronger at longer auditory-cortex response latencies (~300 ms from sound onset). These longer-latency short-term plasticity effects seem to build up more gradually, within tens of seconds after shifting the focus of attention. Importantly, some of the auditory-cortical short-term plasticity effects observed during selective attention predict enhancements in behaviorally measured sound discrimination performance. As so eloquently defined more than a century ago by philosopher William James, selective attention is “the taking possession by the mind, in clear and vivid form, of one out of what seem several simultaneously possible objects or trains of thought. Focalization, concentration, of consciousness are of its essence. It implies withdrawal from some things in order to deal effectively with others, and is a condition which has a real opposite in the confused, dazed, scatterbrained state” . SubsequAs will be reviewed in detail in the following, a number of recent findings have significantly shed light on the neural basis of selective attention. Specifically, it appears that selective attention is supported by short-term plasticity at the level of the auditory-cortex manifested as changes in neuronal receptive fields that filter attended sound features from amongst task-irrelevant ones. While some of these short-term plasticity effects seem to take place very quickly following a shift in the focus of attention, some seem to take longer to build up. Note that in line with our preceding work , 5, we h∼Brodmann area 41 [∼Brodmann area 22). Overall, the nonprimary auditory cortices are more heterogeneous than the primary auditory-cortex, especially in terms of functional anatomy. Nonhuman primate models [While the detailed anatomical subdivisions of human auditory cortex have been more difficult to establish  than area 41 ) and is e models , 30, 31,e models , 33, ande models –36 studie models , 38, loce models –42, and e models , 43.It has been further demonstrated that anterior and posterior nonprimary auditory-cortical areas show greater sensitivity to nonspatial and spatial sound attributes, respectively, in macaque monkeys  and catsRecent research has extended these findings by showing how “top-down” inputs during selective attention reshape the auditory-cortex neuronal receptive fields to help filter relevant stimulus features. In the present review, we focus on describing findings on such short-term plasticity phenomena; specifically how, when, and where top-down inputs modulate sound processing in the auditory system during attentive states, and how such short-term plasticity is associated with enhanced behavioral discrimination ability.One of the most central research questions in the neuroscience of auditory selective attention has been whether selective-attention modulates sound processing already in the primary auditory cortex, or whether the short-term plasticity caused by selective-attention is restricted to nonprimary auditory-cortical areas. Most of the earlier fMRI studies have simply probed whether significant hemodynamic response enhancements can be seen in nonprimary and primary auditory-cortical responses to sounds when they are selectively attended versus ignored. While some of these studies have provided evidence for the predominance of nonprimary auditory-cortex modulations –58, ther∼seconds), the observed modulation of primary auditory-cortex responses could also be due to feedback inputs taking place at longer latencies. Thus, other methods, including electroencephalography (EEG) and magnetoencephalography (MEG), have been used to address the question of at which latencies selective-attention modulates processing of an incoming auditory stimulus.Observing selective-attention effects in primary auditory cortex in fMRI studies does not necessarily imply that selective-attention modulates the initial responses to auditory stimuli within this structure. Especially when using blocked-design paradigms, combined with the relatively low temporal accuracy of the blood-oxygenation level dependent responses that are measured with fMRI  is larg∼50 ms responses has been observed to be significantly enhanced, suggesting that processing of attended sounds is facilitated in primary auditory cortex. The precise mechanisms underlying the enhancement of these early-latency mass-action level responses during selective attention, however, remain an open question.While the vast majority of MEG and EEG studies have documented selective-attention effects at latencies  beyond the initial responses that take place in primary auditory cortex (these findings are reviewed below), there are studies indicating that even the very early responses peaking ~50 ms from sound onset, and estimated to originate in the primary auditory cortex, are augmented by selective attention –67. In tAugmentation of the initial primary auditory-cortex responses by selective attention raises the interesting question of whether the attended auditory stimuli are prioritized already at the level of subcortical auditory nuclei. Anatomically, this would be certainly possible via corticofugal connections that connect corresponding parts of the tonotopic maps, as documented in animal models , 69. OveOverall, it has not been well established to date whether signal enhancements induced by selective attention extend to subcortical auditory structures in addition to auditory cortex in humans. Despite the negative results concerning brainstem auditory evoked potentials –73, evidIt is possible that these discrepancies in findings are explained by the relatively small influence of attention on subcortical processing, combined with the fact that attentional effects depend on the rate of stimulation  and that∼100 ms) has been widely documented, suggesting that selective-attention does induce the most robust short-term plasticity effects in nonprimary auditory-cortical areas. There are fMRI [While there have been relatively few studies describing early-latency selective-attention effects in primary auditory-cortical areas and subcortical structures, modulation of responses originating in nonprimary auditory-cortical structures in slightly longer latencies  sounds along a single sound-feature dimension, it is possible to estimate the average shape of the receptive field of the underlying neural population . The inc∼100 ms from sound onset was best explained by a model combining increased gain and enhanced tuning  recalibration of auditory perception to a new reverberant environment . Sound d∼100 ms response tuning by selective attention and further suggested that the longer-latency ∼300 ms effects develop more slowly, over the time course of several tens of seconds [∼100 ms from stimulus onset are associated more with facilitating rapid shifting of the focus of selective-attention. In contrast, the longer-latency effects could be associated with slower-onset tuning effects, such as those observed in behavioral spatial hearing experiments where gradual adjustment to echo properties of a room have been documented [There have been relatively few studies that have attempted to address the time course of auditory attention in humans. It is possible that the limited signal-to-noise ratios of the temporally accurate EEG and MEG methods available in human studies have limited the number of attempts since the attention shift over multiple trials would have to be repeated tens of times. The results of a recent combined MEG/EEG-fMRI study suggested that nonprimary auditory-cortex neuronal receptive-field changes associated with selective attention take place as rapidly as during the first seconds following shift in the focus of attention . Interes seconds . For an cumented .The question of how quickly the selective-attention effects wear off following withdrawal of attentional focus is related to the question of how quickly the effects develop and can be seen as lingering effects in paradigms where there are alternating shifts in the focus of attention. It has been shown in animal studies that at least some of the receptive-field reshaping effects observed at the level of single primary auditory-cortex neurons linger for extended periods of time after cessation of the task performance . Tentati∼100 ms from sound onset appear to take effect nearly instantaneously, short-term plasticity that modulates processing of sounds at longer latencies seem to build up over much longer time scales of tens of seconds. Given that the short-term plasticity effects predict enhancements in behaviorally measured sound discrimination performance, it can be assumed that auditory-cortex short-term plasticity  underlies the ability of humans to filter the concurrently most relevant stimuli from amongst the countless number of task-irrelevant stimuli. Further research is, however, needed to fully elucidate the relative functional roles of the effects that have been documented to take place during selective attention at the different levels of the human auditory system.It has been shown in both animal models and human neuroimaging studies that selective attention can modulate processing of attended sounds across multiple latencies and at multiple levels of the auditory system. It seems that processing in nonprimary auditory-cortical areas is modulated more robustly during selective attention than in auditory core areas or subcortical auditory structures. Specifically, there is accumulating evidence suggesting that top-down inputs during selective attention stimulus feature specifically reshape the receptive fields of neurons within functionally specialized nonprimary auditory-cortical areas, thus effectively filtering attended sound features from amongst task-irrelevant ones. While the receptive-field reshaping effects that modulate processing at


In the present study, three ionic liquids, namely 1-butyl-3-methylimidazolium chloride ([BMIM]Cl), 1-ethyl-3-methylimidazolium acetate ([EMIM]OAc), and 1-ethyl-3-methylimidazolium diethyl phosphate ([EMIM]DEP), were used to partially dissolve rice husk, after which the cellulose were regenerated by the addition of water. The aim of the investigation is to examine the implications of the ionic liquid pretreatments on rice husk composition and structure.From the attenuated total reflectance Fourier transform-infrared (ATR FT-IR) spectroscopy, X-ray diffraction (XRD) and scanning electron microscopy (SEM) results, the regenerated cellulose were more amorphous, less crystalline, and possessed higher structural disruption compared with untreated rice husk. The major component of regenerated cellulose from [BMIM]Cl and [EMIM]DEP pretreatments was cellulose-rich material, while cellulose regenerated from [EMIM]OAc was a matrix of cellulose and lignin. Cellulose regenerated from ionic pretreatments could be saccharified via enzymatic hydrolysis, and resulted in relatively high reducing sugars yields, whereas enzymatic hydrolysis of untreated rice husk did not yield reducing sugars. Rice husk residues generated from the ionic liquid pretreatments had similar chemical composition and amorphousity to that of untreated rice husk, but with varying extent of surface disruption and swelling.The structural architecture of the regenerated cellulose and rice husk residues showed that they could be used for subsequent fermentation or derivation of cellulosic compounds. Therefore, ionic liquid pretreatment is an alternative in the pretreatment of lignocellulosic biomass in addition to the conventional chemical pretreatments. The increasing demand for lignocellulosic feedstock derived from commodity crops, such as cotton plant and fiber wood crop, has prompted the need to prospect for alternative renewable resources, such as agricultural crop residues. These lignocellulosic crop residues are generated worldwide in vast amounts. In the Asia-Pacific region alone, approximately 1.2 billion tons of crop residues are generated annually OAc showed the highest dissolution of rice husk (Section ‘Dissolution of rice husk’), and its regenerated cellulose possessed all the absorption bands present in the untreated rice husk. This clearly suggests that [EMIM]OAc does not selectively dissolve cellulose, but both cellulose and lignin in the rice husk lignocellulosic matrix. This ionic liquid has been reported to be capable of dissolving cellulose and lignin , and var-1 is broader implying a higher amount of disordered cellulosic structure Cl and [EMIM]DEP.In comparison, the spectra of regenerated cellulose from [BMIM]Cl and [EMIM]DEP dissolution were different from the spectrum of the untreated rice husk, where some absorption bands were absent. In the spectra of regenerated cellulose of these two ionic liquid pretreatments, the band in the region of 800 – 950 cmtructure ,38. The tructure ,37. In aretching . Moreove-1. The transmittance of this band increased in all the rice husk residues compared to their untreated counter-parts. This indicates that the rice husk residues contained considerable amounts of cellulose/hemicellulose, and possibly lignin, after the ionic liquid pretreatments. Besides, this also implies that ionic liquid pretreatment might dissolve components other than cellulose/hemicellulose, whereby the relative cellulose/hemicellulose content of rice husk residues increases proportionately. The intensity of absorption band in the region 800 – 950 cm-1 remains unchanged, signifying that both the rice husk residues and untreated rice husk do not vary very much in terms of amorphousity.The spectra of rice husk residues after ionic liquid dissolution were also recorded using ATR FT-IR Figure . All theθ = 18.7° compared with the untreated rice husk [The crystallinity of the rice husk samples was analyzed by XRD. A less crystalline regenerated cellulose structures was confirmed by XRD analysis with the occurrence of a sharper peak at 2ice husk . The lowice husk . All theRice husk residues of [BMIM]Cl and [EMIM]DEP pretreatments showed higher crystallinity index compared with the untreated rice husk; rice husk residue of [EMIM]OAc pretreatment showed slightly lower crystallinity index that is comparable to the untreated rice husk Table . The disThe structural morphology of cellulose regenerated from the ionic liquids was examined by SEM Figure . All theThe SEM images of the rice husk residues are shown in Figure The quantitative yield of regenerated cellulose reported in ‘Dissolution of rice husk’ indicates only the efficiency of the ionic liquids in dissolving rice husk. Information on structural characterization of the regenerated cellulose as well as the rice husk residues is helpful in the selection of a suitable ionic liquid pretreatment for lignocellulosic biomass. The ATR FT-IR and SEM analyses suggested that regenerated cellulose of the ionic liquid pretreatments comprise of cellulose-rich materials, which were more amorphous compared to the untreated rice husk. According to the XRD analysis, regenerated cellulose of the ionic liquid pretreatments had comparable crystallinity, with cellulose regenerated from [EMIM]DEP and [EMIM]OAc pretreatments showing a lower crystallinity.Apart from the regenerated cellulose, rice husk residues from the ionic liquid pretreatments could be potential substrates for bioconversion into valuable compounds. The disrupted surface structure of the rice husk residues were favorable for solid-state fermentation, where it facilitates microbial growth by allowing access of microbes to the lignocellulosic matrix. SEM investigation demonstrated surface structure disruption of the rice husk residues after ionic liquid pretreatments, whereby [EMIM]OAc-treated rice husk residue showed the highest degree of structural disruption, followed by rice husk residues of [BMIM]Cl and [EMIM]DEP pretreatments. Besides, rice husk residue of [EMIM]OAc was found to have lower crystallinity after pretreatment compared to the other two ionic liquids. Nonetheless, chemical compositions of the rice husk residues remain relatively the same as those of the untreated rice husk.The findings of structural characterization suggested that regenerated cellulose of [EMIM]OAc is amorphous and has low crystallinity, whereas its rice husk residue showed rigorously disrupted structure with reduced crystallinity. Therefore, [EMIM]OAc is a potential ionic liquid for the pretreatment of rice husk.To confirm the digestibility of the regenerated cellulose from ionic liquid pretreatment, enzyme hydrolysis of the regenerated cellulose were carried out. Figure Besides acid and alkaline pretreatments, ionic liquid pretreatment can be used for pretreating lignocellulosic biomass. This study found that the chemical composition of the regenerated cellulose varies with the type of ionic liquid used. The ionic liquids [BMIM]Cl and [EMIM]DEP delignified the lignocellulosic rice husk, indicating their potential to be used in producing regenerated cellulose for enzymatic saccharification or cellulose derivatives. On the other hand, [EMIM]OAc dissolved the entire lignocelluloses and imparted surface structure disruption on the regenerated cellulose that is desired for subsequent fermentation or derivation of cellulosic compounds. The regenerated cellulose were more amorphous and had lower crystallinity compared with the untreated rice husk, whereas the rice husk residues showed a certain degree of structural disruption. The study also demonstrated that enzymatic hydrolysis of the regenerated cellulose resulted in higher yield compared to the untreated rice husk. Both the regenerated cellulose and rice husk residue revealed desirable structural changes in this study, which suggested that ionic liquid pretreatment is beneficial for conversion into value-added products. It is also essential to select a suitable ionic liquid pretreatment depending on the final application of the regenerated cellulose and rice husk residue.Rice husk samples were obtained from Selangor, Malaysia. The rice husk was first washed and dried before being ground to approximately 30 mesh sizes (500 μm). Ground rice husk samples were stored in a dry cabinet prior to use.The ionic liquid 1-butyl-3-methylimidazolium chloride ([BMIM]Cl) was purchased from Merck (Germany), while 1-ethyl-3-methylimidazolium acetate ([EMIM]OAc) and 1-ethyl-3-methylimidazolium diethyl phosphate ([EMIM]DEP) were purchased from Sigma-Aldrich (USA). The ionic liquids were used without further purification. Their chemical structures are illustrated in Figure Trichoderma viride  was purchased from Merck (Germany). The CMC activity of the Cellulase Onozuka R-10 was reported to be ≥ 1 U/mg. The IUPAC Filter Paper Assay was determined according to the procedure outlined by Ghose Cl: 1-butyl-3-methylimidazolium chloride; [EMIM]DEP: 1-ethyl-3-methylimidazolium diethyl phosphate; [EMIM]OAc: 1-ethyl-3-methylimidazolium acetate; ATR FT-IR: Attenuated total reflectance Fourier transform-infrared; SEM: Scanning electron microscopy; XRD: X-ray diffraction.The authors declare that they have no competing interests.GCN, ASMC, and MGL (supervisors) conceived the study. TNA carried out the pretreatment experiments and analyses, and drafted the manuscript. GCN and ASMC participated in the test design and supervision, and helped to draft the manuscript. MGL provided ideas on the methodology and proofread the manuscript. All authors read and approved the final manuscript.