Attention deficit hyperactivity disorder (ADHD) presents special challenges for drug development. Current treatment with psychostimulants and nonstimulants is effective, but their mechanism of action beyond the cellular level is incompletely understood. We review evidence suggesting that altered reinforcement mechanisms are a fundamental characteristic of ADHD. We show that a deficit in the transfer of dopamine signals from established positive reinforcers to cues that predict such reinforcers may underlie these altered reinforcement mechanisms, and in turn explain key symptoms of ADHD. We argue that the neural substrates controlling the excitation and inhibition of dopamine neurons during the transfer process are a promising target for future drug development. There is a need to develop animal models and behavioral paradigms that can be used to experimentally investigate these mechanisms and their effects on sensitivity to reinforcement. More specific and selective targeting of drug development may be possible through this approach.The online version of this article (doi:10.1007/s13311-012-0132-y) contains supplementary material, which is available to authorized users. Attention deficit hyperactivity disorder (ADHD) is a behaviorally defined disorder of unknown etiology and pathophysiology. As such, it presents special challenges to develop animal models for drug development. In this review, we consider evidence that altered mechanisms for positive reinforcement may be a fundamental characteristic of ADHD and suggest that sensitivity to positive reinforcement is a useful construct for animal models and paradigms for research into new treatments for ADHD. We have not undertaken a comprehensive review because a number of thorough reviews have recently appeared in the literature dealing separately with animal models –8, reinfFor clarity in terminology, we will use the term “positive reinforcer,” or simply “reinforcer” to refer to a stimulus that increases the likelihood of an associated response, or a stimulus that would elicit an approach reaction, commonly referred to as a reward. We use the term “reinforcement learning” to refer to the associative process that occurs when a behavior is followed by a positive reinforcer. In humans, the term “primary reinforcer” is too restrictive for the reinforcing events that motivate behavior, which include social reinforcers, such as praise or money. For these events, we use the term “established positive reinforcer.” We first review behavioral aspects of ADHD and evidence for altered positive reinforcement mechanisms. We then review the neurobiology of positive reinforcement and the central role of the neurotransmitter dopamine in reinforcement learning. We consider a postulated deficit in the transfer of dopamine signals from established positive reinforcers to cues that predict positive reinforcers as an explanation for altered reinforcement sensitivity in ADHD. Next we consider rodent models for ADHD that have been evaluated in relation to reinforcement, and their potential use in drug development. This includes a review of behavioral paradigms that can be used to assess sensitivity to reinforcement. We conclude that few models have been adequately tested for altered reinforcement sensitivity, or for their ability to predict the effectiveness of new compounds for treatment of ADHD, particularly altered reinforcement sensitivity. Finally, we consider new directions for the development of animal models that could test the effects of novel therapeutic agents on dopamine responses to cues and established positive reinforcers.ADHD is a common and debilitating disorder diagnosed on the basis of persistent and developmentally inappropriate levels of over-activity, inattention, and impulsivity . The symRather than trying to model ADHD in a categorical way, an argument can be made for identifying component behavioral characteristics. For example, impulsivity is a component of ADHD. The ability of a drug to reduce impulsivity may predict therapeutic efficacy in ADHD, even if it does not affect other symptoms. This may be tested in a normal rat or in an animal expressing levels of impulsivity lying at the extreme of the normal range.Clinical reports, together with a growing number of experimental studies, have identified differences in the manner in which children with ADHD respond to reinforcement . HistoriSeveral studies report differences between children with ADHD and controls in the effects of reinforcement on cognitive task performance  hypothesis as an explanation for altered processing of positive reinforcement in children with ADHD , 93. TheIn ADHD, we propose the transfer of the dopamine response to previously neutral cues is disrupted. In particular, we assume that the phasic dopamine cell response to cues that predict reinforcement is reduced in amplitude to the point of being ineffective . In addition, we assume there is a failure of the mechanism that inhibits dopamine cell firing at the time of the primary reinforcement. The net result of these 2 assumptions is a failure of the dopamine signal to transfer to the earlier cues that predict reinforcement. Instead, the dopamine signal continues to occur at the time of established positive reinforcers. Moreover, if the reinforcer is intermittent or delayed, the dopamine signal is likewise intermittent and delayed. We argue as follows that this may cause key symptoms of ADHD.The exact nature and cause of the failure is currently unknown. However, there are many ways that such failure might occur. For example, altered dopamine transporter function, or altered dopamine receptor function, as a consequence of genetic polymorphisms in these molecular mechanisms, could disrupt the transfer process. Other possibilities also exist, such as abnormalities in the systems that are afferent to the dopamine cells, which may include prefrontal cortical mechanisms. The precise cause of the deficient transfer, however, is a separate matter from the symptoms that can be predicted from it.We predict that such failure would lead to a number of symptoms of ADHD. As a consequence of failure to predict reinforcers, individuals with ADHD experience a delayed dopamine signal (delayed reinforcement), or no signal (discontinuous reinforcement) at the cellular level, rather than the immediate anticipatory dopamine signal experienced by typically developing individuals. This would make them abnormally sensitive to delayed or discontinuous reinforcement. It would also have the effect of making individual instances of actual reinforcement more salient, which may result in greater control by incidental stimuli in the environment over the integrated history of reinforcement. These corollaries are developed more fully in earlier theoretical articles , 93, 96.The assumption that dopamine signaling in humans parallels that measured in experimental animals has some support from functional magnetic resonance imaging (fMRI) studies. Changes in blood flow and oxygenation measured by fMRI signals are thought to provide an indirect measure of dopamine release. In particular, dopamine may activate postsynaptic neurons by potentiation of corticostriatal synapses, and therefore increase local fMRI signals . FunctioRecent functional studies in humans have been enhanced by the use of models based on the temporal difference algorithm for predicting reinforcement. This machine-learning algorithm generates prediction error signals that correlate with dopamine signals in primates during Pavlovian conditioning . In humaversus those with ADHD during gain anticipation with a monetary incentive delay task. These studies are consistent with the assumption of reduced response to cues.Evidence from recent fMRI studies provides support for the DTD hypothesis of impaired anticipatory dopamine cell firing in ADHD. In normal subjects, preference for immediate in comparison with delayed reinforcement is associated with the magnitude of ventral striatal activity . AdolescEvidence for the assumption that dopamine cells continue to fire in response to established positive reinforcers is more equivocal. Strohle et al.  observedThere are several ADHD symptoms that can be interpreted as arising from a dysfunction in the transfer of dopamine cell firing. Symptoms of inattention include: “often fails to give close attention to details or makes careless mistakes in schoolwork, work, or other activities”; “often has difficulty sustaining attention in tasks or play activities” and “is often easily distracted by extraneous stimuli.” These symptoms occur with higher frequency in children with ADHD, and may be explained by impaired anticipatory dopamine release. The reason for this is that if the dopamine release occurs only in response to actual instances of reinforcement, incidental aspects of the environment that activate dopamine neurons may reinforce off-task behaviors rather than the socially established cues present in classroom situations. Other symptoms of inattention can be interpreted as failure of conditioned reinforcers leading to increased sensitivity to delay of reinforcement and less effective performance under schedules of partial reinforcement. These include: “often does not follow through on instructions and fails to finish schoolwork, chores, or duties in the workplace,” and “often avoids, dislikes, or is reluctant to engage in tasks that require sustained mental effort.”The DTD theory also explains some symptoms of hyperactivity and impulsivity. The symptom of “often leaves seat in classroom or in other situations in which remaining seated is expected” may be interpreted as a lack of effective reinforcement for remaining seated arising from a deficit in anticipation of reinforcement. The symptoms “often has difficulty awaiting turn,” “often blurts out answers before questions have been completed,” and “often interrupts or intrudes on others” involve a delay between the target behavior and the actual reinforcement. Therefore, they may be interpreted as being due to abnormal sensitivity to delay of reinforcement arising from a failure of the transfer of dopamine cell firing activity in response to cues that predict reinforcement.Although many ADHD symptoms can be explained by DTD, it would be possible to have DTD and not meet criteria for ADHD, because some symptoms might arise by different mechanisms. However, DTD does provide a distinct framework for the development of animal models and new targets for pharmacological compounds for a particular set of symptoms.Because the neurotransmitter dopamine is involved in the processing of reinforcement and reinforcement learning, it is an important system in the development of new medications to treat ADHD. In particular, the importance of timing requirements suggests that ADHD medications should target anticipatory release of dopamine. However, the primary action of the drugs currently used in the treatment of ADHD is an action on dopamine signals, which is not specific to the timing or cause of the dopamine signal. They may compensate for a deficit in dopamine transfer, but do not correct the primary deficit of failure to transfer. Such specificity of action may not be possible with drugs that act on the dopamine transporter rather than the circuitry that controls the dopamine neurons.The molecular actions of existing drugs used in the management of ADHD provide limited insight into the underlying pathology of ADHD. It is commonly assumed that because these drugs are indirect dopamine agonists, there must be an overall deficiency of dopamine. However, no such overall deficiency has been conclusively identified, and more subtle alterations in dopamine function, such as DTD may be important for the development of new variants of these drugs and novel therapeutic compounds. Currently 2 major classes of drugs are used in the treatment of ADHD: 1) psychostimulants and 2) norepinephrine reuptake inhibitors. Amphetamine  and methylphenidate  are psychostimulants. Atomoxetine (Strattera) is a norepinephrine reuptake inhibitor.At therapeutic dosages, an amphetamine is a dopamine reuptake inhibitor and also a norepinephrine reuptake inhibitor, and it is also a very weak inhibitor of serotonin reuptake . At highAtomoxetine (Strattera) is a nonstimulant that acts as a presynaptic blocker of norepinephrine reuptake , increasVolkow et al.  showed tHuman studies support suggestions that actions on reinforcement mechanisms underlie the therapeutic effects of methylphenidate. In children with ADHD, methylphenidate has been shown to raise the breakpoint  on progressive ratio tasks , 123. MeA number of different rodent models have been proposed, and there are several comprehensive reviews , 126. HeThe symptoms of ADHD are not unique to it and are present in some degree in the normal population. Similarly, psychostimulant drugs modify attention, impulsivity, and hyperactivity in people with and without ADHD. The use of “normal” rats, with appropriate behavioral measures, is thus well justified. A number of excellent reviews of behavioral paradigms are available  is a rat version of the continuous performance test used in humans . In thisThe 5-CSRT can be used to identify rats within a “normal” population with a deficit in selective attention accompanied by impulsivity. Rats selected for high levels of impulsivity on a 5-CSRT task also exhibited high levels of impulsive decision-making on a delay-of-reinforcement task . HoweverPuumala et al.  used a 5Stop-signal reaction time tasks (SSRTs) measure the ability to withhold or inhibit an already initiated or pre-potent motor response . Stop-siThe spontaneously hypertensive rat (SHR) is the most widely used animal model of ADHD –149. SHRClosely related to the delay-discounting paradigm, Sutherland et al.  adapted . [. [There have been relatively few studies of the effects of psychostimulants on the SHR. In the T-maze task, previously described, in which rats had to choose between a small, but immediate and a large, but delayed reinforcer procedure , the SHR.  found th. [.  investig. [.  investig. [. .Similar to the SHR, a genetically independent, hypertensive rat strain, known as the Genetically Hypertensive (GH) rat, was developed in New Zealand by selective breeding of Wistar rats for hypertension –159. TheThe GH provides an interesting complement to the SHR, in that in both strains the hyperactivity has arisen from the selection for high blood pressure, although is not related to blood pressure per se. This convergence across strains suggests that the relevant genes may be physically close, but they are not identical to those for hypertension. However, further work is needed to analyze the behavioral characteristics of the GH rat.Neurochemically, selective lesions of the dopamine neurons by neonatal administration of the neurotoxin 6-hydroxydopamine (6-OHDA) lead to locomotor hyperactivity , 164. ThA 6-OHDA mouse model has also been developed, which shows increased activity levels and a reduction in activity after treatment with psychostimulants , 172. ThSeveral pieces of evidence suggest that abnormal dopamine transporter (DAT) function may be important in ADHD. Overexpression of DAT has been found in human ADHD, and 1 of the major actions of the psychostimulants is to block DAT. Thus, DAT dysfunction has been proposed as a possible cause of ADHD, and mice with genetically engineered changes of DAT function have been proposed as models for use in ADHD research –175. ForBased on the increasing evidence that altered reinforcement mechanisms may underlie or be associated with some symptoms in ADHD , we suggThe DTD hypothesis identifies certain neural targets for intervention in ADHD. In particular, the circuitry mediating excitation of dopamine neurons by cues predicting reinforcement is a potential target. Candidate structures include the basolateral amygdala projection to the nucleus accumbens,  central The identification of the neurobiological substrates underlying reinforcement by basic research suggests more specific and selective targeting of drug development may be possible, focusing on the mechanisms that mediate anticipation of reinforcement. The therapeutic effects of existing drugs can be understood in this framework, but there are likely to be improvements within their wide spectrum of action on neuromodulators. For example, blocking dopamine transporter activity probably increases dopamine signals evoked by both cues and established positive reinforcers. According to the DTD hypothesis, a better result would be to increase the dopamine signal in response to the cues, and decrease the signal in response to the established positive reinforcer. To achieve this it would be necessary to target the neural circuitry that excites the dopamine neurons in response to the cues (and enhance its effects), and separately target the circuitry that inhibits the dopamine neurons at the time of the reinforcer. To date, we know of no treatment designed to produce these effects.Advances in understanding the pathophysiology of ADHD in terms of altered reinforcement sensitivity may also lead to the development of animal models of specific components of the ADHD syndrome. This may range from the selection of phenotypes from normal populations, selective breeding, genetic manipulation, or specific lesions. Another possibility is optogenetic inhibition of dopamine cell firing at the time of cue presentation, directly simulating DTD.With these advances, it may be possible to target specific symptoms of ADHD for selective treatment. It is well known that ADHD is a heterogeneous disorder that overlaps with other behavioral disorders. Treatments that target reinforcement mechanisms are unlikely to cover all aspects of the disorder. However, such treatments may reduce specific symptoms or processes that affect learning and socialization.Disclosure forms(PDF 510 kb)Below is the link to the electronic supplementary material.


WRAP53 regulates p53 expression and WRAP53 protein is a component of telomerase. NATs play key roles in carcinogenesis, and although WRAP53 is known to increase cancer cell survival, its role in ESCC clinicopathology is unknown. The aim of this study was to investigate WRAP53 expression in ESCC and to correlate it with clinicopathological characteristics.Esophageal squamous cell carcinoma (ESCC) is a highly aggressive cancer whose underlying molecular mechanisms are poorly understood. The natural antisense transcript (NAT) WRAP53 mRNA and protein expression was measured by quantitative PCR (qRT-PCR) and western blotting, respectively, in 4 ESSC cells lines and in 45 paired ESCC and non-neoplastic esophageal mucosa tissues. To correlate WRAP53 protein expression with clinicopathological characteristics, immunohistochemistry (IHC) was performed on 134 ESCC and 85 non-neoplastic esophageal mucosa tissues.Expression of WRAP53 was detected in all ESCC cell lines and was upregulated in the ESCC tissues compared with the corresponding non-neoplastic tissues (P<0.01). More cells expressed WRAP53 protein in the ESCC tissues than in the non-neoplastic tissues (P<0.01). Overexpression of WRAP53 was significantly correlated with tumor infiltration depth (P = 0.000), clinical stage (P = 0.001), and lymph node metastasis (P = 0.025). Wrap53 expression was not correlated with age, gender, or tumor differentiation.This report indicates increased expression of WRAP53 in ESCC and that WRAP53 overexpression is correlated with tumor progression. WRAP53 may play a significant role in ESCC; accordingly, WRAP53 could be a useful biomarker for ESCC. Antisense RNAs may also encode proteins or may exist only as non-protein-coding transcripts WRAP53 regulates endogenous p53 mRNA levels and therefore has a critical role in p53 function. Overexpression of WRAP53 increases p53 mRNA and protein levels WRAP53 mRNA also encodes WRAP53 protein , which has been identified as essential for Cajal body maintenance by binding and directing small Cajal body-specific RNAs (scaRNAs) to the Cajal bodies Esophageal cancer (EC) is one of the most common malignant tumors, resulting in poor prognosis worldwide In this study, we investigated the expression of WRAP53 protein and mRNA in EC cell lines, ESCC tumors, and adjacent non-neoplastic esophageal mucosa tissue. Our work indicates that WRAP53 is overexpressed in ESCC tissue compared to adjacent non-neoplastic esophageal mucosa tissue and that WRAP53 expression closely correlates with the clinicopathology in ESCC patients.This study was approved by the Ethics Committee of Shantou University Medical College. During this study, informed consent in writing was obtained from each patient and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki as reflected in a priori approval by the Ethics Committee of Shantou University Medical College.The esophageal carcinoma cell lines KYSE150 and KYSE180 were kindly provided by professor Liyan Xu Tissues were obtained from 134 patients  who had undergone radical esophagectomy in the Department of Thoracic and Cardiovascular Surgery, Second Affiliated Hospital of Shantou University Medical College  from 30 March, 2008, to 15 July, 2011. Forty-five pairs of samples were preserved in liquid nitrogen immediately after collection for subsequent testing. Each sample was matched with the adjacent non-neoplastic mucosa removed during the same surgery, usually 5–10 cm away from the periphery of the main tumor lesion. All patients were selected at their first diagnosis and none had received radiotherapy, chemotherapy, and/or immunotherapy before the esophagectomy. All ESCC and adjacent non-neoplastic mucosa tissues were independently confirmed by two pathologists who were blinded to the original diagnosis. For this examination, strict criteria were used to diagnose the non-neoplastic mucosa tissue as having no carcinoma, dysplasia, or atypical hyperplasia; however, chronic inflammation was allowed for inclusion. We collected clinical data, including gender and age of patients, depth of tumor invasion, cell differentiation, lymph node metastasis, and clinical tumor-node-metastasis (TNM) stage. Primary tumor staging followed the seventh edition of the TNM staging system of the American Joint Committee on Cancer (AJCC). Depth of infiltration was classified into four groups as follows: pT1 to submucosa, pT2 to muscularis propria, pT3 to adventitia, and pT4 to adjacent structures Immunohistochemical staining with anti-WRAP53 antibody  was performed as follows: tissues were fixed in 10% formaldehyde, embedded in paraffin, cut into 4-μm sections, and mounted on slides. Slides were deparaffinized, rehydrated, and antigen unmasking was processed in 0.01 M sodium citrate buffer (pH 6.0) at high temperature (120°C) for 5 min, cooled at room temperature for 30 min, and immersed in 3% hydrogen peroxide solution for 10 min. Slides were washed twice in PBS, blocked with 10% normal goat serum at 37°C for 30 min, and then incubated with rabbit polyclonal WRAP53 antibody overnight at 4°C. After washing with phosphate-buffered saline (PBS), the sections were treated with corresponding streptavidin peroxidase–conjugated secondary antibody. Diaminobenzidine (DAB) was used to visualize WRAP53 antibody binding, and the tissue sections were counterstained with hematoxylin. Primary antibody was replaced by PBS in the negative control.Immunostaining for WRAP53 was graded by a semiquantitative method on a scale that took into account the intensity and distribution of the staining. WRAP53 immunostaining was examined by two pathologists using light microscopy. WRAP53 expression was determined from at least 1,000 cells that were systematically counted at ×400 magnification in five visual fields. In the immunohistochemistry (IHC) assay for WARP53, the presence of nuclear staining was considered to be significant. Nuclear staining was detected in all tissue samples examined, whereas cytoplasmic staining was detected in some, but not all tumor samples. We therefore measured and quantified WRAP 53 staining within the nucleus. Staining intensity was graded as 0 (negative), 1 (weak), 2 (moderate), and 3 (strong); the percentage of positive cells examined was scored as 0 (no positive cells), 1 (< 10%), 2 (11–50%), 3 (51–80%), and 4 (> 80%). The two scores were multiplied and the immunoreactive score  was determined: 0 (negative), 1–3 (weak), and 4–6 (positive); multiplication values of 8, 9, and 12 were scored as strongly positive WRAP53 and GAPDH gene sequences reported in the literature and as available in GenBank WRAP53 primers were the following: forward, 5′-TGGCACAAAGCTGGACAGT-3′ and reverse, 5′-GCTGGGTCCTGGTCTGAAG-3′. The GADPH primers were the following: forward, 5′-GCACCGTCAAGGCTGAGAAC-3′ and reverse, 5′-TGGTGAAGACGCCAGTGGA-3′. The specificity of the amplification was confirmed through dissociation curve analysis yielding single peaks from PCR products; 2% agarose gel electrophoresis was used to confirm the correct sizes of the PCR products. GAPDH expression was used as an internal control to normalize WRAP53 expression in the samples. PCR reactions of each sample were conducted in triplicate. The relative expression of WRAP53 was calculated by the 2−ΔΔCt method (ΔΔCt  = (CtWRAP53-CtGAPDH)ESCC tissue-(CtWRAP53-CtGAPDH)matched non-neoplastic esophageal mucosa tissue. For the matched non-neoplastic esophageal mucosa tissue control sample, the ΔΔCt was 0 and 2−ΔΔCt was 1.Total RNA was isolated from 45 pairs of frozen tissue samples  using TRIzol reagent . The concentration and purity of the RNA in each sample was measured by absorbance at 260 and 280 nm using a spectrophotometer. Total RNA was reverse transcribed into single-stranded cDNA by using the RT reagent Kit . Real-time quantitative PCR was performed with an ABI Prism 7000 (Applied Biosystems) machine and by using Platinum SYBR Green qPCR SuperMix-UDG . The PCR amplification consisted of the following cycling program: 50°C for 2 min, 95°C for 2 min, followed by 40 cycles of 95°C for 15 s and 60°C for 30 s. The PCR primer sequences were designed according to the human g for 15 min at 4°C. Proteins from the different groups were separated by 10% SDS–PAGE and transferred to polyvinylidene difluoride membranes  at 100 V for 120 min on ice. The membranes were blocked with 5% non-fat milk in Tris-buffered saline with Tween 20 , followed by incubation with a rabbit polyclonal antibody for WRAP53 (1∶1000 in TBST) or rabbit polyclonal antibody for β-actin (1∶2500 in 5% nonfat milk in TBST) overnight at 4°C. After 3 washes in TBST, membranes were exposed to horseradish peroxidase–conjugated secondary antibody  for 1 h at room temperature. Proteins were detected by enhanced chemiluminescence  and exposed to X-ray film. β-actin was used as a loading control. Protein concentrations were determined with a Gel-pro Analyzer 4.0 . WRAP53 protein expression was calculated from the relative intensity ratio of WRAP53 to β-actin protein.Tissues selected by the qRT-PCR method were subjected to protein analysis by using western blotting. Frozen tissues were lysed in RIPA buffer  containing a protease-inhibitor cocktail on ice for 30 min. The different groups of esophageal carcinoma cells were washed 3 times with PBS before lysis. The resulting cell lysates were clarified by centrifugation at 12,000 ×All statistical analyses were performed with SPSS 13.0 for Windows , and the data were expressed as the mean ± standard deviation (SD). The Wilcoxon test was used to evaluate the statistical significance of the difference in the expression of WRAP53 mRNA and protein. The Chi-square test was used to determine correlations between WRAP53 expression and clinicopathological parameters. In all analyses, a P<0.05 was considered statistically significant.To investigate the relationship between WRAP53 expression and ESCC, we first examined the expression of WRAP53 in 4 ESCC cell lines  we detected the expression of WRAP53 protein in all 4 ESCC cell lines with an expected molecular weight of 75 kDaFigure 1WRAP53 mRNA expression in the ESCC tissues and in the adjacent non-neoplastic esophageal mucosal tissues. WRAP53 mRNA expression was upregulated in 37/45 (82.2%) of the esophageal carcinoma specimens. The qRT-PCR analysis also showed that the levels of WRAP53 mRNA expression in ESCC tissues were significantly higher than those in the corresponding non-neoplastic esophageal mucosal tissues (P<0.01)  Esophageal cancer (EC) is one of the most common malignant diseases worldwide. The processes during EC carcinogenesis and progression involve complex factors, stages, and changes at the molecular level WRAP53 mRNA expression was upregulated in 82% of the ESCC patients. Taken together, these results suggest that WRAP53 may act as an oncogene in ESCC. WRAP53 protein expression in EC tissues was significantly higher than that in non-neoplastic esophageal mucosa tissues (P<0.001). As far as we know, this is the first report showing a difference of WRAP53 expression in cancerous and non-cancerous tissues. Therefore,WRAP53 expression could be involved in ESCC development.The WRAP53 gene has been shown to be involved in the development of primary human cancer. Schildkraut et al. have found that some single-nucleotide polymorphisms (SNPs) in WRAP53 modestly increase the risk of serous and endometrioid invasive ovarian cancer We also characterized the expression pattern of the WRAP53 protein by using (IHC). The IHC results showed that, in general, WRAP53 was predominantly expressed in the nuclei of tumor cells, especially in immature tumor cells and progenitor cells in the basal and/or suprabasal layer. These immunohistochemical studies suggest that WRAP53 might be involved in the proliferation of ESCC. Investigations with samples from patients with head and neck squamous cell carcinoma showed that WRAP53 levels are higher in patients with recurrent tumors compared with patients with positive cancer outcomes In conclusion, our results have shown that the expression of WRAP53, the natural antisense transcript to p53, is significantly upregulated at the level of both mRNA and protein in ESCC tissues compared with non-neoplastic esophageal mucosa tissues. Overexpression of WRAP53 correlated with tumor infiltration depth, clinical stage, and lymph node metastasis. Therefore, WRAP53 may play a significant role in the development and progression of ESCC. Thus, WRAP53 could be a useful biomarker for ESCC and could represent a potential target for treatments of this disease.


Ex vivo voltammetry allows for the determination of ethanol effects on presynaptic dopamine terminals, independent of drug-induced changes in firing rates of afferent inputs from either dopamine neurons or other neurotransmitter systems. However, differences in striatal dopamine dynamics did not predict the locomotor-activating effects of ethanol, since the inhibitory effects of ethanol on dopamine release were similar between strains. There were differences in presynaptic dopamine function between strains, with faster dopamine clearance in the caudate-putamen of DBA/2J mice; however, it is unclear how this difference relates to locomotor behavior. Because of the role of the dopamine system in reinforcement and reward learning, differences in dopamine signaling between the strains could have implications for addiction-related behaviors that extend beyond ethanol effects in the striatum. A large body of research has aimed to determine the neurochemical factors driving differential sensitivity to ethanol between individuals in an attempt to find predictors of ethanol abuse vulnerability. Here we find that the locomotor activating effects of ethanol are markedly greater in DBA/2J compared to C57BL/6J mice, although it is unclear as to what neurochemical differences between strains mediate this behavior. Dopamine elevations in the nucleus accumbens and caudate-putamen regulate locomotor behavior for most drugs, including ethanol; thus, we aimed to determine if differences in these regions predict strain differences in ethanol-induced locomotor activity. Previous studies suggest that ethanol interacts with the dopamine transporter, potentially mediating its locomotor activating effects; however, we found that ethanol had no effects on dopamine uptake in either strain.  DBA/2J (DBA) and C57BL/6J (C57) mice are two inbred strains that show disparate phenotypes with respect to ethanol preference, drinking, and reward, among many other ethanol-mediated behaviors -6. The s In addition to the differences in drinking behavior and ethanol reward between DBA and C57 mice, DBA mice are more sensitive to the locomotor-activating effects of ethanol , althougex vivo voltammetry is that it allows for the determination of the effects of ethanol on striatal terminals, independent of afferent inputs from both the dopamine system and other neurotransmitter systems. This technique is particularly useful as many pharmacological approaches to developing treatments for psychiatric disorders rely on an understanding of the region-specific effects of drugs. Because it has been demonstrated that ethanol’s ability to increase dopamine levels is due to a balance of its actions on dopamine terminals [ The primary focus of the current research was to assess ethanol-induced locomotor activity in DBA and C57 mice, and whether or not this activity was mediated by altered dopamine dynamics at the level of striatal terminals. DBA mice exhibited much greater locomotor activation by ethanol, an effect that was specific to ethanol, since other locomotor stimulating events elicited opposite responses. For example, DBA mice exhibited reduced locomotor activation in a novel environment compared to C57 mice, as shown here and by others . In ordeerminals  and moduerminals -15, thisad libitum. Brain slices from both strains were obtained from naïve animals after at least one full week of habituation to the housing colony. The Institutional Animal Care and Use Committee at Wake Forest University School of Medicine approved the experimental protocol (Protocol Number: A10-177). All mice were cared for according to the National Institutes of Health guidelines in Association for Assessment and Accreditation of Laboratory Animal Care accredited facilities. Male DBA and C57 mice  were used for all experiments. Animals were group housed in polycarbonate cages and maintained on a 12:12 light-dark cycle (7:00 pm lights off) with standard rodent chow and water Locomotor activity was assessed via infrared beam breaks in automated locomotor activity monitors . Mice (n=9-10 per strain) were first placed into activity monitor chambers for 120 minutes to record response to novelty and to allow for habituation to the chamber. Twenty-four hours following habituation, animals were placed in the same chambers for 30 minutes before administration of two injections of saline, 60 minutes apart, to desensitize the animals to injection stress and allow for a within-subject control. Every day thereafter, animals received ethanol injections  in the locomotor chambers after 60 minutes of habituation. Activity in response to each injection was reported for the first 30 minutes. Locomotor activity was measured as distance traveled (in centimeters) and as a percent of each animal’s second saline injection.Fast scan cyclic voltammetry in brain slices was used to characterize pre-drug striatal dopamine kinetics, as well as the effects of quinpirole and ethanol on single pulse evoked dopamine release. Briefly, a vibrating tissue slicer was used to prepare 400 µm thick coronal brain slices containing the striatum. Slices were incubated in oxygenated artificial cerebrospinal fluid and heated to 32°C. A carbon fiber microelectrode  and a bipolar stimulating electrode  were placed in close proximity (≈100 µM) on the surface of the slice, in either the NAc core or CPu. Endogenous dopamine efflux was induced by a single, rectangular, electrical pulse applied every five minutes for four milliseconds . Dopamine release was detected by applying a triangular waveform  every 100 milliseconds to the recording electrode. Background current subtraction methods were applied to obtain clear current versus time plots. When baseline collections were stable for three consecutive stimulations, quinpirole  or ethanol  was bath applied cumulatively to brain slices. Electrodes were calibrated immediately following experiments by recording their response (in nA) to 3μM dopamine using a ﬂow-injection system.  To determine dopamine release and clearance, representative signals were analyzed before bath application of quinpirole or ethanol. Dopamine release (µM) was calculated as the amount of dopamine released per electrical stimulation whereas clearance was determined using the rate constant, tau. All data was collected and analyzed with Demon Voltammetry and Analysis software .o-38°C for 15 minutes. Immediately following incubation, the plate was analyzed with SoftMax Pro Software version 5 . Blood ethanol concentrations (BEC) from DBA and C57 mice were obtained at 5, 15, 30, 45, 60, 90 and 120 minutes after a 0.5 g/kg i.p. dose of ethanol . After injection, a submandibular vein blood draw was performed at each of the respective time points and blood was collected in BD microtainer tubes lined with lithium heparin . Each animal had no more than two bilateral blood draws per day and blood collection volumes did not exceed the maximum set forth by the institutional Animal Care and Use Committee. For determination of BECs, standards and samples were prepared with a commercially available alcohol dehydrogenase assay . Briefly, five microliters of each blood collection was placed in a container with 45μl of trichlorocetic acid solution  and centrifuged at 10,000 revolutions per minute at room temperature for ten minutes. 30μl of the supernatant of each sample was removed and placed into a separate container with 300μl of buffer and 112.5μl of enzymatic solution provided in the alcohol dehydrogenase assay. 100μl of each standard and sample was loaded, in triplicate, into a 96 well plate, covered and incubated at 37Statistical analyses and graphs were prepared using Graph Pad Prism . Summed data for the groups’ locomotor response to novelty and saline injection as well as baseline release and tau were compared across groups using a two-tailed Student’s t-test. Locomotor data (in time bins) and voltammetric data (quinpirole and ethanol concentration response curves) were compared using a two-way analysis of variance (ANOVA) with strain and dose as factors, and BECs were compared using a two-way ANOVA with strain and time as factors. When a significant main effect was obtained (p < 0.05), Bonferroni post-hoc analysis was used to determine significant effects.1, 19 = 14.68, p < 0.001; p < 0.05) and 0.5 (p < 0.01) g/kg doses. Analysis revealed no difference between DBA and C57 mice, with respect to their locomotor response to a saline injection, indicating that basal locomotor activity did not differ between the two strains. Because DBA and C57 mice do not differ in their responses to a saline injection, their differential behavioral responses to ethanol cannot be attributed to disparate baseline locomotor activity levels. To study the behavioral sensitivity of DBA and C57 mice to ethanol, we determined ethanol-induced locomotor activity over a range of ethanol doses (0.125-2.0 g/kg). Two-way ANOVA revealed a main effect of strain on ethanol-induced locomotor behavior . Furthermore, we found an effect of time on locomotor response to novelty  and a novelty x strain interaction .To determine if the enhanced response to ethanol in DBA mice was specific to ethanol or was due to an enhanced response to all locomotor-activating stimuli, we examined locomotor responses to a novel environment in DBA and C57 mice. Two-way ANOVA revealed a main effect of strain on response to novelty . However, we found no differences between strains with respect to blood ethanol elimination rate at any time point tested , where quinpirole dose-dependently reduced evoked dopamine release. The effects of quinpirole on dopamine release were similar between strains, demonstrating that D2-like autoreceptor function was not different . Next, we aimed to determine if differences between strains were present in the CPu. We found that DBA and C57 mice have similar dopamine release in the CPu , left; h4,13 = 41.17, p < 0.001), where quinpirole significantly decreased dopamine release over increasing concentrations of the compound. We found no strain differences in autoreceptor function as both strains had similar sensitivity to the effects of quinpirole  and CPu , where increasing concentrations of ethanol significantly reduced evoked dopamine release. We found that DBA and C57 mice have similar responses to bath-applied ethanol in both the NAc core (To determine if increased ethanol-induced locomotion in DBA mice is mediated by ethanol effects on striatal dopamine terminals, we examined the effects of ethanol at the terminal by bath application of increasing doses of the drug over brain slices. A two-way repeated measures ANOVA revealed a significant effect of ethanol in both the NAc core (FNAc core  and CPu NAc core . In addition to determining the effects of ethanol on evoked dopamine release, we also determined the effects of ethanol on dopamine clearance. Dopamine clearance is mediated by the DAT, thus changes in the clearance of dopamine following bath application of ethanol can give information as to how ethanol alters DAT function. Contrary to previously published reports, we showed no effect of ethanol on dopamine clearance. Further, DBA and C57 mice have similar synaptic dopamine clearance measures in the NAc core  and CPu in vivo models following ethanol administration are most likely not due to the effects of ethanol on dopamine terminals. Here we show that, although ethanol-induced locomotor activity is enhanced in DBA versus C57 mice, this difference is not due to ethanol’s effects on dopamine release or DAT activity. The ability of ethanol to reduce stimulated dopamine release in the NAc core and CPu was similar between the two strains of mice. Also, evoked dopamine release and uptake were comparable between the two strains, except in the CPu, where DBA mice exhibited faster clearance. Although some work has suggested that ethanol has direct effects at the DAT, here we show that ethanol does not influence dopamine uptake via the DAT in either the NAc core or CPu. Previous microdialysis work has shown that ethanol increases dopamine levels in the striatum to a greater extent in DBA mice , and thiThe enhanced response to ethanol in DBA mice as compared to C57 mice is not due to an enhanced response to all locomotor-activating stimuli, as DBA mice showed a reduced response to novelty. Responses to novelty have been shown previously to correlate with acquisition of stimulant self-administration and addiction vulnerability for these compounds -26. Accoin vivo increases in dopamine overflow, and could explain the locomotor differences between the two strains. In addition, postsynaptic dopamine receptors, which have been shown to have differential expression levels between the two strains, may play a role in the behavioral disparities [Although differences in locomotor activity point to differential striatal dopamine system functioning between DBA and C57 mice, our data indicate that these effects do not occur at the level of the dopamine terminal. It has been shown previously that DBA mice have enhanced ethanol-induced dopamine overflow, an effect that is likely mediating the enhanced ethanol-induced locomotion in this strain . Electroparities . in vivo preparation have found ethanol-induced decreases in dopamine uptake in the olfactory tubercle, an effect that could lead to increased dopamine levels following ethanol administration [in vivo, it is possible that ethanol effects on other neurotransmitters systems are involved in modulating dopamine dynamics, including uptake. Ex vivo voltammetry allows for the isolation of dopamine terminals separate from afferent inputs, which allows for the determination of ethanol effects directly at the DAT. Here we show that ethanol does not affect dopamine clearance by direct interactions with the DAT. Although previous research has pointed to the DAT as being altered by acute ethanol exposure -18, it rstration . However Here we demonstrate that ethanol-induced modulations of dopamine release and clearance at the level of the striatum are not mediating ethanol-induced locomotor activity. We demonstrate that the dopamine release inhibiting effects of ethanol do not differ between strains, and that ethanol does not have any direct effects at the DAT. Furthermore, our data adds to a body of literature showing that the effects of ethanol on the dopamine system are a balance of its inhibitory and excitatory effects. We show here that ethanol, when applied to the dopamine terminal, results in reduced stimulated release, while previous work has shown that ethanol, when bath applied to VTA cell bodies, results in enhanced firing. It is likely that these effects converge to result in the behavioral outputs that are observed following ethanol administration.


The ability to microdissect individual cells from the nervous system has enormous potential, as it can allow for the study of gene expression in phenotypically identified cells. However, if the resultant gene expression profiles are to be accurately ascribed, it is necessary to determine the extent of contamination by nontarget cells in the microdissected sample. Here, we show that midbrain dopamine neurons can be laser-microdissected to a high degree of enrichment and purity. The average enrichment for tyrosine hydroxylase (TH) gene expression in the microdissected sample relative to midbrain sections was approximately 200-fold. For the dopamine transporter (DAT) and the vesicular monoamine transporter type 2 (Vmat2), average enrichments were approximately 100- and 60-fold, respectively. Glutamic acid decarboxylase (Gad65) expression, a marker for GABAergic neurons, was several hundredfold lower than dopamine neuron-specific genes. Glial cell and glutamatergic neuron gene expression were not detected in microdissected samples. Additionally, SN and VTA dopamine neurons had significantly different expression levels of dopamine neuron-specific genes, which likely reflects functional differences between the two cell groups. This study demonstrates that it is possible to laser-microdissect dopamine neurons to a high degree of cell purity. Therefore gene expression profiles can be precisely attributed to the targeted microdissected cells. The midbrain dopamine system, comprising the nigrostriatal, mesocortical, and mesolimbic pathways, is involved in many brain functions, such as motor control, cognition, and reward behaviors . DysfuncThere is considerable cellular heterogeneity within the substantia nigra (SN) and VTA regions of the midbrain. Stereological estimates of GABA and dopamine neuron populations in SN and VTA indicate that there are more GABA than dopamine neurons, and in some subregions there are nearly three times as many . Additioα-synuclein transcript levels in SN dopamine neurons from PD brains [Recent studies have demonstrated the potential of dopamine neuron microdissection. For example, Liss and colleagues were able to distinguish midbrain dopamine neuron subpopulations on the basis of tyrosine hydroxylase (TH), vesicular monoamine transporter type 2 (Vmat2), and dopamine transporter (DAT) transcript expression ratios [D brains . Such coD brains . The molD brains . FurtherD brains , reliablWe have previously reported a method that permits the extraction of high quality RNA from immunolabeled, microdissected cells . The priAll animal work was carried out in strict accordance with institutional, state, and national animal ethics regulations. Tissue preparation and identification of midbrain dopamine neurons by TH immunofluorescence was carried out essentially as described . BrieflyμL RLT lysis buffer  in lots of 100. Following cell collection, lysis buffer volume was increased to 100 μL, and the sample homogenized by vortexing and then placed at −80°C for later use. Prior to RNA extraction, the three 100 dopamine neuron samples were combined, and total RNA was extracted using the RNeasy microkit (Qiagen) according to manufacturer's instructions, including addition of poly-A carrier RNA to the lysate and an on-column DNAse-I treatment.Laser microdissection and pressure catapulting of dopamine neurons were carried out using a PALM MicroBeam system (Zeiss). Briefly, TH immunolabeled midbrain dopamine neurons were visualised with the system's Axiovert 200 M inverted fluorescent microscope. The UVA laser was then focused in the plane of the tissue section, and the cell body was separated from surrounding tissue using laser ablation to remove a thin line of tissue at the cell body border. Removal of the cell body from the glass slide was accomplished through defocussing the laser beam and pressure catapulting cells into lysis buffer contained in a PCR tube lid, mounted directly above the cells of interest. For both laser cutting and catapulting, the minimum amount of laser energy was used to achieve separation of the cell from its surrounding tissue (laser cutting) and for removal of the cell from the microscope slide (laser catapulting). For each animal a total of three hundred dopamine cell bodies from each of the SN and VTA were UVA laser microdissected. To minimize the length of time tissue sections were held on the microdissection microscope stage, and cells were pressure catapulted into 30 μL RT+ cDNA synthesis reaction, with the remaining third used in the RT− reaction. One microliter of cDNA product was added to each qPCR reaction, with the qPCR protocol, and all other reagents and volumes as described [Reverse transcription was carried out using SuperScript III (Invitrogen), according to manufacturer's instructions and as previously described . Two-thiescribed . Primer escribed  was usedμL RT+ and RT− cDNA synthesis reactions and 1 ng of cDNA added to each qPCR reaction.To enable assessment of dopamine neuron enrichment conferred by microdissection, total RNA from whole midbrain sections (containing dopamine neurons) was extracted, contaminating genomic DNA removed by on-column DNase-I digestion using Qiagen's RNeasy microkit and DNase reagents according to manufacturer's instructions. The concentration of total RNA was determined using Thermo Scientific NanoDrop 1000 spectrophotometer. Reverse transcription and qPCR were carried out as described above, with 60 ng of RNA added to 20 t-test for each gene. A comparison of the SN and VTA dopamine neuron molecular profiles was also statistically analyzed using an unpaired Student's t-test. An alpha level of .05 was applied for all statistical tests.To assess purity and enrichment of the microdissected population, a comparison between whole midbrain sections and either SN or VTA laser microdissected dopamine neurons was made using an unpaired Student's As is evident from To determine the level of purity of the microdissected cells, relative expression levels for dopamine-related and nondopamine-related genes were quantified. The dopamine neuron-specific genes were those encoding the dopamine transporter (Slc6a3/DAT), tyrosine hydroxylase (TH), vesicular monoamine transporter type 2 (Slc18a2/Vmat2), and the nuclear orphan receptor (Nr4a2/NURR1). The nondopamine neuron-specific genes were the glutamate neuron-specific vesicular glutamate transporter type 2 (Slc17a6/Vglut2), the GABA neuron-specific glutamic acid decarboxylase 65 (Gad2/Gad65), and the glial cell-specific glial fibrillary acidic protein (Gfap). The relative expression levels of all dopamine neuron-related transcripts were significantly increased in the SN and VTA microdissected dopamine neurons compared to whole sections, with DAT being 104- and 95-, TH 147- and 249-, Vmat2 60- and 57-, and NURR1 4- and 10-fold enriched in SN and VTA, respectively . NeitherP < 0.01) and DAT expression significantly higher than Vmat2 (P < 0.01), and this was the case for both SN and VTA. The relative expression for each gene between the SN and VTA was then compared. P < 0.01), there was no significant difference in the DAT/Vmat2 ratio between the two regions (P = 0.57), and there was a trend toward a higher TH/Vmat2 ratio in the VTA (P = 0.06).The midbrain SN and VTA dopamine neuron populations are, for the most part, anatomically separate. It is possible, therefore, to microdissect the dopamine neurons of the two populations and carry out a comparative analysis. Firstly, we compared expression levels of the dopamine neurotransmission-related genes  within each of the two populations and for both the SN and VTA. TH expression was significantly higher than DAT (The ability to microdissect specific cells from complex, heterogeneous tissues, such as the brain, is advantageous for transcriptomics as it limits a potential artifact, caused by dilution effects, that is characteristic of studies using tissue homogenates as starting material. However, if meaningful interpretations of transcriptomic investigations of specific cell types or single cells are to be obtained, two important issues need to be considered. These include sample RNA integrity and the degree of contamination with untargeted cells or cell fragments. We have previously developed and demonstrated a method that preserves RNA integrity during the immunolabeling process that is generally obligatory for the identification of cells for microdissection . In the To assess the level of enrichment for dopamine neurons in microdissected samples, dopamine neuron-associated gene expression was compared to that in whole midbrain sections. The degree of enrichment reflects the relative number of target and nontarget cells within the section being microdissected and the efficiency of the microdissection process in terms of its ability to quantitatively capture all the targeted cells. As predicted, expression levels of the dopamine neuron-specific genes, DAT, TH, and Vmat2, and the dopamine neuron-associated gene NURR1, were all found to be markedly increased, relative to the 18S reference gene, in microdissected dopamine neurons compared to whole midbrain sections, and this was the case in both the SN and VTA. In comparison to DAT, TH, and Vmat2, the enrichment of NURR1 was modest, presumably reflecting its lack of absolute specificity for dopamine neurons in the midbrain , 11. ImpTo be able to accurately attribute changes in gene expression to a particular cell or cell type, it is important to determine the purity of the sample. Contamination of microdissected dopamine neuron samples by glial cell, glutamate, and GABA neurons was assessed by qPCR for the presence of Gfap, Vglut2, and Gad65 transcripts, respectively. Transcripts for either Gfap or Vglut2 were not detected in microdissected dopamine neuron samples, indicating that contamination by either glial or glutamate cells did not occur to a significant level. However, a small but significant contamination by GABA neurons was found, with Gad65 transcripts being detected in microdissected dopamine neuron samples from both the SN and VTA. The level of GABA transcript contamination was extremely low, being approximately 500-fold less than the average for dopamine neuron-specific TH transcripts in the SN, and it was not reliably detected in all samples as the qPCR Ct value was typically close to 40. The level of GABA neuron contamination was higher in the VTA samples, which could have resulted from an increased difficulty to microscopically visualize, select, and extract often smaller more clustered VTA dopamine neurons during the laser microdissection process [Our lack of evidence for Vglut2 transcripts in microdissected dopamine neuron samples is somewhat surprising given the neuroanatomical , 12, 13,One advantage of the immunolaser-microdissection technique used in the present study is that it can precisely isolate individual cells. Therefore, it was possible to accurately collect the anatomically segregated dopamine neurons of the SN and VTA. It is well established that dopamine neurons of the VTA and SN and subpopulations within these nuclei are similar with respect to the dopamine phenotype, but distinct with regard to other properties, such as their ability to regulate calcium , respondBy combining an immunofluorescent labeling method with laser microdissection to specifically target midbrain dopamine neurons, this study demonstrates that large numbers of these neurons can be extracted from separate cell groups to produce samples enriched and relatively pure for SN and VTA dopamine neurons. Indeed, this method could readily be combined with existing tract tracing techniques to allow for greater dopamine neuron subpopulation analysis. As the method employed an immunolabeling protocol designed to preserve high quality RNA, it also allows for the meaningful interpretation of downstream molecular analysis.


Ankylosing spondylitis is a chronic inflammatory disease characterized by inflammatory lower back pain and morning stiffness and accompanied by spine and sacroiliac joint involvement. Klinefelter's syndrome is a genetic condition that only affects males. Affected males have an extra X chromosome. This paper reports a 30-years-old male on followup with the diagnosis of Klinefelters syndrome. The patient admitted with complaints of inflammatory lower back, and neck pain and morning stiffness and was diagnosed with ankylosing spondylitis. Nonsteroidal anti-inflammatory drug and salazopyrine treatment resulted in significant regression in his complaints. Klinefelters syndrome (KS) is a genetic disease affecting only males, in which there is an extra X chromosome. A male or a boy diagnosed with KS has 3 sex chromosomes 47,XXY)  or 46,XY/47XXX (mosaic form) instead of usual one X and one Y chromosomes  . Various7,XXY (cl30-years-old male patient has been on followup by the endocrinologist for about 20 years with the diagnosis of KS . The patEndocrine system and gender dysmorphism play an important role in the development, maintenance, and regulation of immune response. In females, response to antigenic stimuli and increased prevalence of autoimmune disease are more common. These observations are perceived as the evidence of regulation of immune response by gender dysmorphism via thymic-hypothalamus-pituitary-gonadal axis . The coeInformation on the coexistance of KS and AS in the literature is limited , 9. In aIn conclusion, we have reported a rare case of AS and KS coexistance. The existence of extra X chromosome in KS may play an important role in AS expression. Besides, used in the treatment of KS, testosterone may play a part in triggering and/or severe progression of AS. Further studies are required to highlight the pathogenesis of autoimmune diseases in KS.


Escherichia coli synthesizes three anaerobically inducible [NiFe]-hydrogenases (Hyd). All three enzymes have a [NiFe]-cofactor in the large subunit and each enzyme also has an iron-sulfur-containing small subunit that is required for electron transfer. In order to synthesize functionally active Hyd enzymes iron must be supplied to the maturation pathways for both the large and small subunits. The focus of this study was the analysis of the iron uptake systems required for synthesis of active Hyd-1, Hyd-2 and Hyd-3 during fermentative growth.feoB gene encoding the ferrous iron transport system. The levels of both hydrogen-oxidizing enzymes Hyd-1 and Hyd-2 as determined by specific in-gel activity staining were reduced at least 10-fold in the mutant after anaerobic fermentative growth in minimal medium, while the hydrogen-evolving Hyd-3 activity was less severely affected. Supplementation of the growth medium with ferric iron, which is taken up by e.g. the siderophore enterobactin, resulted in phenotypic complementation of the feoB mutant. Growth in rich medium demonstrated that a mutant lacking both the ferrous iron transport system and enterobactin biosynthesis (entC) was devoid of Hyd-1 and Hyd-2 activity but retained some hydrogen-evolving Hyd-3 activity. Analysis of crude extracts derived from the feoB entC double null mutant revealed that the large subunits of the hydrogen-oxidizing enzymes Hyd-1 and Hyd-2 were absent. Analysis of lacZ fusions demonstrated, however, that expression of the hya, hyb and hyc operons was reduced only by maximally 50% in the mutants compared with the wild type.A transposon-insertion mutant impaired in hydrogenase enzyme activity was isolated. The mutation was in the feoB entC double mutant and this appears to be due to post-translational effects. The retention of residual hydrogen-evolving activity, even in the feoB entC double null mutant suggests that sufficient iron can be scavenged to synthesize this key fermentative enzyme complex in preference to the hydrogen-uptake enzymes.Our findings demonstrate that the ferrous iron transport system is the principal route of iron uptake for anaerobic hydrogenase biosynthesis, with a contribution from the ferric-enterobactin system. Hydrogen-oxidizing enzyme function was abolished in a  Escherichia coli encodes four membrane-associated [NiFe]-hydrogenases, only three of which are synthesized under standard anaerobic growth conditions. Two of these enzymes, hydrogenase 1 (Hyd-1) and Hyd-2, oxidize hydrogen while the third, Hyd-3, is part of the hydrogen-evolving formate hydrogenlyase (FHL) complex -hydrogenases catalyze the reversible activation of molecular hydrogen . The genemainder . This pr- are attached [The active site of the [NiFe]-hydrogenases comprises a Ni atom and a Fe atom to which the diatomic ligands CO and CNattached . The Hypattached ,5. Iron attached . In addiattached ,8.E. coli has been well characterized -hydrogenases during fermentative growth in null mutants lacking defined iron transport systems.While the route of nickel transport for hydrogenase biosynthesis in cterized ,9, it haous iron . Under at system ,12. Undeuisition . These FE. coli catalyze the hydrogen-dependent reduction of the artificial redox dye benzyl viologen (BV) -hydrogenases in gen (BV) ,14. Thisgen (BV) . Approxinsporter .feoB::Tn5) grown anaerobically in M9 minimal medium with glucose as carbon source and with different iron sources (Table -1 (Table 2+) resulted in extracts with a reduced but still significant hydrogen-oxidizing activity of 66% compared to the wild type grown without addition. It was noted that due to the poor growth of the strains in minimal medium in the presence of ferricyanide and ferrocyanide the hydrogenase enzyme activity was highly variable with high SD values. This phenomenon was reproducibly observed, despite attempts to harvest cells under strictly comparable conditions of growth and presumably reflects variability in the labile Hyd-3 activity (see below). Therefore, it must be stressed that only general trends in enzyme activity changes caused by these iron sources can be considered.The hydrogen-dependent reduction of BV was determined in extracts derived from MC4100 (wild type) and PM06 . The plasmid failed to restore hydrogenase enzyme activity to the levels determined for the wild type; surprisingly, the presence of the plasmid reduced overall hydrogenase activity to only about 15% that of the wild type (Table In an attempt to complement the pe Table . WesternfeoABC operon on the plasmid restored hydrogenase activity in PM06 to wild type levels (Table feoB gene caused a polar effect on the downstream feoC gene and only the presence of the complete operon on a plasmid could complement the mutation.Introduction of the complete ls Table . This laentC) or the uptake system for ferric-citrate  essentially had little to no effect on total hydrogenase activity . Combining the entC and fecA-E mutations (strain CP415) reduced hydrogenase activity by approximately 60% compared to the wild type. Introducing the feoB::Tn5 mutation into this strain to deliver CP413 (entC fecA-E feoB::Tn5) reduced total hydrogenase activity even further such that only approximately 7% of the wild type level could be detected.Single null mutations that prevented biosynthesis of ferric-enterobactin (strain CP416 Δty Table . IntroduentC) and CP422 (fecA-E) showed essentially wild-type Hyd-1 and Hyd-2 activity profiles  showed clearly reduced intensity bands for both enzymes, which is in accord with the results after growth in minimal medium  or CP413 (entC fecA-E feoB::Tn5) grown fermentatively in rich medium had neither Hyd-1 nor Hyd-2 enzyme activities. This result indicates that the residual hydrogenase enzyme activity in CP413 must result from Hyd-3 (compare Table entC feoB::Tn5) had an FHL activity of approximately 50% of the wild-type, strain CP413 (entC fecA-E feoB::Tn5) still retained 30% of the wild-type FHL activity, confirming that the residual hydrogenase activity in extracts of CP413 was indeed due to Hyd-3.Analysis of cell-free extracts derived from these strains grown fermentatively in rich medium by non-denaturing PAGE, with subsequent staining for activity of Hyd-1 and Hyd-2, revealed that, as anticipated, the extracts of CP416  and CP422 (fecA-E) both showed levels of processed large subunits for Hyd-1, Hyd-2 and Hyd-3 similar to those seen for the wild-type MC4100  and CP413 (entC fecA-E feoB::Tn5), on the other hand, essentially completely lacked either the unprocessed or processed forms of the large subunits of Hyd-1 or Hyd-2, which correlates with the lack of Hyd-1 and Hyd-2 enzyme activity observed in Figure In order to determine whether the lack of Hyd-1 and Hyd-2 activity in the mutants devoid of ferrous and ferric uptake was due to lack of processing of the large subunits because of iron-limitation, the precursor and processed forms of the large subunits of Hyd-1, Hyd-2 and Hyd-3 in cell-free extracts derived from the iron transport mutants after growth in rich medium were analysed by Western blotting with enzyme-specific antisera. Extracts derived from MC4100 (wild type) revealed mainly the processed form of the catalytic subunit of all three enzymes Figure , which it Figure , which i0 Figure . Densito0 Figure . Extracthya, hyb and hyc operons encode Hyd-1, Hyd-2 and Hyd-3, respectively [lacZ translational fusions to the first gene of each operon, which encode the respective small subunits of the enzymes Hyd-1 and Hyd-2, while the hycA gene encodes a transcriptional regulator (see Methods). After transfer to the lambda phage λRS45 [hyaA'-'lacZ, hybO'-'lacZ and hycA'-'lacZ fusions were introduced in single copy onto the chromosome of the respective mutants. To demonstrate that the fusions were functional we analyzed expression levels after growth under both aerobic and anaerobic conditions. Expression of hyaA'-'lacZ was strongly reduced when wild type cells were grown aerobically, while expression was up-regulated approximately 70-80 fold during fermentative growth .The results in Table E. coli was significantly affected by introduction of a fur mutation. Fur is a global regulator controlling iron homeostasis [fur mutant [fur mutation, which has both negative and positive effects on gene expression of iron metabolism including depression of iron uptake systems, caused a strong reduction in FHL activity, suggesting Fur is required for FHL synthesis. In the current study we could show in an otherwise Fur+ background that causing iron limitation by removing key iron uptake systems also resulted in differential effects on hydrogen uptake and hydrogen evolution: hydrogen-oxidizing hydrogenase function was compromised first while hydrogen-evolving hydrogenase activity was partially retained. During a search for genes affecting hydrogenase biosynthesis or activity, a mutant with a transposon insertion in feoB encoding the GTPase component of the postulated ferrous iron transport system [2+ ions are available and the Feo transport system is active [In a previous study  it was seostasis ,25. Diffr mutant . The furt system  was isols active -12, it wfeoB mutation with a mutation in entC, which impairs biosynthesis of the siderophore enterobactin, abrogated Hyd-1 and Hyd-2 activities. Notably, however, significant Hyd-3, and consequently FHL, activity was retained in the double null mutant, suggesting that when iron is limited during fermentative growth the synthesis of the hydrogen-evolving Hyd-3 takes precedence over the two hydrogen-oxidizing enzymes Hyd-1 and Hyd-2. The fact that Hyd-2 is maximally active under more reducing conditions, while Hyd-1 is an oxygen-tolerant enzyme and is active at more positive redox potentials [zupT and mntH mutations were also introduced into this background was FHL activity abolished [Combining the tentials , did notbolished . This suhyp mutants, where active site biosynthesis cannot be completed [lacZ fusions of the hya and hyb structural gene operons was largely unaffected by the deficiency in iron transport suggests that a different level of regulation in response to iron availability exists. This regulation might possibly be post-translational, for example through altered protein turnover due to insufficient iron.A somewhat unexpected result of this study was the finding that under iron limitation no unprocessed species of the Hyd-1 or Hyd-2 large subunits were present and only very low amounts of the processed proteins were observed. This was unexpected because in ompleted , signifihya, hyb or hyc operons. The Hyd-3 component of the FHL complex was less severely affected by defects in these iron uptake systems, indicating that a greater degree of redundancy in iron acquisition for this enzyme exists. Thus, when iron becomes limiting during fermentative growth synthesis of active Hyd-3 has priority over that of the hydrogen-oxidizing enzymes Hyd-1 and Hyd-2. This probably reflects a physiological requirement to maintain an active FHL complex to offset acidification of the cytoplasm caused by formate accumulation via disproportionation of the metabolite into the freely diffusible gaseous products CO2 and H2.Mutants unable to acquire iron through the ferrous iron transport and siderophore-based uptake systems lacked the hydrogen-oxidizing enzymes Hyd-1 and Hyd-2 under anaerobic fermentative conditions. Iron limitation did not affect transcription of the All bacterial strains, plasmids and phages used in this study are listed in Table E. coli was grown aerobically in Erlenmeyer flasks filled to maximally 10% of their volume with LB medium on a rotary shaker (250 rpm) and incubated at 37°C. Anaerobic growths were performed at 37°C in sealed bottles filled with anaerobic medium and under a nitrogen gas atmosphere. Cultures for determination of hydrogenase processing or for enzyme activity measurements were grown either in buffered TGYEP medium  pH 6,5 [3 was omitted from SLA and was replaced by the appropriate iron source at the concentration indicated. Dipyridyl was added at a final concentration of 300 μM. All growth media included 0.1 μM NiCl2. The antibiotics kanamycin, ampicillin, and chloramphenicol, when required, were added to the medium at the final concentrations of 50, 100, and 12.5 μg per ml, respectively. When indicated anhydrotetracycline (AHT) was added at the final concentration of 0.2 μg per ml.For the purposes of chromosomal and plasmid DNA isolation, ) pH 6,5  suppleme) pH 6,5  containi) pH 6,5  except whyaA and hybO were constructed by amplifying the respective promotor regions and the nucleotides coding for the first 14 or 13 amino acids, respectively, by PCR using Phusion DNA polymerase  and the oligonucleotides hya_regulat_up 5'-GCG GGA TCC GCG CAG AGA TTC GAA CTC TG-3', hya_regulat_down 5'-GCG GGA TCC TGA CGC CGC ATG GCC TGG TA-3', hybO_-217 5'-CTC GGA TCC TAT GGC CGG TTA TCG CCT C-3' and hybO_+38 5'-CTC GGA TCC ATG CCG TGA GAA TGG ATG A-3'. The resulting respective 565 bp and 274 bp fragments were digested with BamHI and ligated into pRS552 [hycA'-'lacZ fusion was constructed using plasmid pTL101 [hyaA'-'lacZ), Φ(hybO'-'lacZ) and Φ(hycA'-'lacZ) protein fusions were introduced in single copy into the lambda attachment site of the respective mutants as indicated in Table The translational fusions to o pRS552 , which ho pRS552 . In a sid pTL101 . The resori/KAN-2 > transposome  atmosphere  and kanamycin-resistant colonies were analysed via a soft-agar overlay technique with benzyl viologen (BV) at a final concentration of 0.5 mM and in a hydrogen atmosphere as described [kc according to Miller [XL1-Blue (Stratagene) was mutagenized using the < R6Kγologies; ) accordiescribed . Colonieo Miller  and the In order to determine the transposon insertion site, chromosomal DNA was isolated from the mutants , digeste600 nm of approximately 0.8. Cells from cultures were harvested by centrifugation at 4,000 × g for 10 min at 4°C, resuspended in 2-3 ml of 50 mM MOPS pH 7.0 buffer and lysed on ice by sonication (30 W power for 5 minutes with 0.5 sec pulses). Unbroken cells and cell debris were removed by centrifugation for 15 min at 10, 000 × g at 4°C and the supernatant was used as the crude cell extract. Protein concentration of crude extracts was determined [M value of 8,600 M-1 cm-1 was assumed for reduced benzyl viologen. One unit of activity corresponded to the reduction of 1 μmol of hydrogen per min. Formate hydrogenlyase (FHL) activity was measured according to [Anaerobic cultures were harvested at an ODtermined  with bovtermined  except trding to  using gaBeta-galactosidase assay was performed in microtiter plates according to  using a Aliquots of 50 μg of protein from crude cell extracts were separated on 10% (w/v) SDS-polyacrylamide gel electrophoresis (PAGE)  and tranNon-denaturating PAGE was performed using 7.5% (w/v) polyacrylamide gels pH 8.5 and included 0.1% (w/v) Triton-X100 in the gels . SamplesWhere indicated, the relative intensity of hydrogenase staining and protein amount from immunoblots was quantified using ImageJ from the National Institutes of Health .Hydrogenase activity-staining was done as described in  except tThe authors declare that they have no competing interests.CP carried out the experimental studies and helped draft the manuscript. GS conceived and coordinated the study and drafted the manuscript. Both authors read and approved the manuscript.feoB::Tn5)Plasmid-encoded FeoB synthesis in MC4100 and PM06 (. Extracts (25 μg protein in membrane sample buffer) from MC4100 and PM06, transformed with pECD1079 bearing feoB and pFEO bearing the whole feo operon, both cloned behind a tetracycline promotor and encoding an N-terminal StrepII-tag on FeoB encoded on pECD1079 were separated by SDS-PAGE (10% w/v polyacrylamide) and after transfer to nitrocellulose detected by incubation with Strep-tactin conjugated to horseradish peroxidase. Strains were grown either with or without aeration in TGYEP, pH 6.5 and gene expression was induced with 0.2 μg ml-1 AHT (anhydrotetracycline) as indicated. Biotin carboxyl carrier protein (BCCP) served as a loading control. The sizes of the protein standards are shown on the right side of the gel. The angled arrow indicates the position of the Strep-FeoB polypeptide. Extracts derived from MC4100 and PM06 transformed with pFEO did not synthesize Strep-tagged FeoB and therefore acted as a negative control.Click here for file


The physical effort required to seek out and extract a resource is an important consideration for a foraging animal. A second consideration is the variability or risk associated with resource delivery. An intriguing observation from ethological studies is that animals shift their preference from stable to variable food sources under conditions of increased physical effort or falling energetic reserves. Although theoretical models for this effect exist, no exploration into its biological basis has been pursued. Recent advances in understanding the neural basis of effort- and risk-guided decision making suggest that opportunities exist for determining how effort influences risk preference. In this review, we describe the intersection between the neural systems involved in effort- and risk-guided decision making and outline two mechanisms by which effort-induced changes in dopamine release may increase the preference for variable rewards. Foraging animals must often consider the variability or risk associated with resource delivery. For example, the preference for variable as opposed to stable sources of food can change dramatically based on a number of factors, such as an animal's current energetic state. In the case of food delivery, animals can express a preference for variable or “risky” food sources when the energetic demands of foraging are high Caraco, . Juncos,To our knowledge, no investigation into the neural basis for the influence of energetic state or physical effort on risk preference has been performed. Consequently, almost nothing is known regarding the neural basis for interactions between effort and risk. Opportunities do exist to explore this question given recent advances in the study of the neural basis of effort- and risk-guided decision making  results in a degree of effort-avoidance that is similar to the degree observed following bilateral ACC or BLA lesions (Floresco and Ghods-Sharifi, Interactions between the ACC and the NAC also appear to influence effort-guided behaviors. For example, the excitotoxic disconnection of the ACC and NAC core also results in effort-avoidance on a barrier-climbing task (Hauber and Sommer, In agreement with the proposed role of the ACC in action selection (Shima and Tanji, It must be cautioned that while these observations highlight similarities between primates and rodents, such comparisons are complicated by notable anatomical and functional differences between species Preuss, . For exaDopamine is traditionally viewed as a key modulator of reward-driven learning; however, the observation that dopamine modulates effort-guided behaviors suggests that this view requires elaboration Wise, . For exaThe influence of dopamine on effort depends on dopamine's effect on specific structures within the effort-network. For instance, targeted 6-OHDA depletion of dopaminergic terminals or blockade of dopamine receptors within the NAC (Salamone, Although there is clear evidence for a role of dopamine release in the NAC in effort-guided behaviors, virtually nothing is known regarding how this release is regulated. For example, the principal source of dopaminergic innervation of the NAC is the VTA, and it is unknown what systems drive VTA activity during effort-guided behaviors. The ACC could be involved given its capacity to trigger burst-firing in dopaminergic neurons (Gariano and Groves, Dopamine can modulate the activity of its targets across many time scales Schultz, , and theLittle is known regarding the relative contribution of phasic and tonic release to effort-guided behaviors; however, three recent studies using fast scan cyclic voltammetry were unable to identify a clear effect of effort on phasic responses within the NAC (Day et al., The variable effects of dopamine on effort-guided behaviors and the different timescales at which dopamine influences neural activity present significant challenges to researchers hoping to create a unifying theory of function. These challenges are compounded by experimental results that suggest that the involvement of dopamine and the NAC in effort-guided behaviors depends on the specific form of physical effort being employed. For example, 6-OHDA depletion of NAC dopamine significantly impairs effort-guided behavior when effort is assessed using fixed-ratio schedules; however, the same dopaminergic manipulation has no effect when effort is in the form of a weighted lever (Ishiwari et al., Studies in humans, non-human primates, and rodents have identified diverse cortical and subcortical structures involved in setting the preference or aversion to risk. In human studies, the Iowa Gambling Task has become an established task for investigating the neural basis for risk-guided decision making (Bechara et al., A criticism of the use of the Iowa Gambling Task for assessing risk-preference is that the task places multiple cognitive demands upon subjects that are unrelated to risk. This is problematic as a preference for risky alternatives or neural responses related to risk preference could, in this task, result from a reduced capacity to shift attention, to perform behavioral reversals, to process losses, or to store items in working memory (Maia and McClelland, Connecting results from human studies to results from studies using non-human primates and rodents is a significant challenge given the wide range of structures identified within and across species. Even so, there are points of intersection. Interestingly, many of these points of intersection include elements of the effort network, such as the ACC, NAC, BLA, and dopamine (Amiez et al., Multiple lines of evidence suggest a role of the primate ACC in evaluating risk and in altering risk preference. In non-human primates, the spiking activity of subsets of ACC neurons changes monotonically with the probability that a cue predicts reward (Kennerley et al., When compared to results from primates, results from studies in rodents have not produced consistent evidence for a role of the ACC in risk-guided behaviors. Although inactivation (St. Onge and Floresco, Dopamine plays a central albeit complex role in modulating the preference for risk. For example, systemic administration of dopamine D1 and D2 antagonists in rats results in the avoidance of high-risk/high-reward options in a 2-lever risk-discounting task (St. Onge and Floresco, Results from single unit-studies of dopaminergic neurons in the primate VTA also suggest a role of dopamine in evaluating risk. For example, the activity of dopaminergic neurons follows the probability of receiving a future reward with the activity of these neurons peaking when the capacity to predict a reward is lowest (Fiorillo et al., increases risk preference when reward probability is high (Norbury et al., As in effort-guided decision making, the influence of dopamine on risk preference may be through dopamine's regulation of the NAC. For example, inactivation or lesions of the NAC in rodents reduces the preference for large, uncertain rewards (Cardinal and Howes, Taken together, these results suggest that increased NAC dopamine enhances the tolerance of risk while reductions in NAC dopamine reduces risk preference. A similar pattern was observed for effort, with enhanced NAC dopamine resulting in an increased willingness to work for rewards and reduced dopamine resulting in effort avoidance. The following section presents possible mechanisms by which these similar patterns contribute to the influence of effort on risk preference.The BLA is also associated with the assessment of risk, although fewer studies have targeted the BLA relative to the regions discussed previously. For example, results from a study by Ghods-Sharifi et al.  suggest Increased metabolic demands and expenditure of physical effort can enhance the preference for variable rewards Caraco, . It has Dopamine is an important modulator of both effort- and risk-guided behaviors. Here we propose that the release of dopamine in the NAC in response to physical effort enhances risk preference. This idea is supported by the following observations. First, dopamine concentrations within the NAC increase with physical exercise (Freed and Yamamoto, What remains to be specified is the mechanism by which NAC dopamine concentration is regulated during effort-guided behaviors. In this regard, the ACC may play a central role given abundant evidence for ACC involvement in effort-guided behaviors (Reviewed in section Experimental Approaches for Studying Effort-Guided Behaviors, Cortical and Sub-Cortical Circuits Involved in Effort-Guided Behaviors and Dopamine and Effort-Guided Behavior and see Figure The capacity of the ACC to sustain responses over delays may be particularly important for maintaining effortful behaviors as such behaviors often require maintaining motor responses over seconds and minutes. Consequently, understanding the timescale of dopamine release in the NAC during effortful behavior may be critical for understanding its role in modulating risk-preference. As discussed in section Dopamine and Effort-Guided Behavior, dopamine release can occur on multiple timescales, and the timescale most relevant for effort- and risk-guided behaviors has yet to be determined, although evidence is building that phasic patterns of dopamine release in the NAC are not critical for effort-guided behaviors (Gan et al., Interestingly, a recent study has identified within-trial increases in striatal dopamine concentration that ramps up as rats approach goal locations on a T-maze (Howe et al., The preceding account suggests that effort-induced changes in NAC dopamine enhance risk-preference and provides the motivational signal required for animals to sustain effortful behaviors. An alternative mechanism by which effort could influence risk is through dopamine's impact on stimulus-reward learning. Dopamine has an established role in facilitating such learning (Schultz et al., An interesting feature of this idea is that it may account for the paradoxical capacity of effort to enhance the reinforcing properties of cues and primary rewards. Pigeons, for example, prefer colored lights, spatial locations, and primary rewards associated with high effort (Clement et al., The recent trend of using ethologically motivated experimental design to connect field observations with observations from neuroscience (Glimcher, The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.