Moreover, while the NO synthase activity was inhibited by doxorubicin-induced ROS, the NO synthase inhibition did not affect doxorubicin-induced ROS. The inhibition of NO synthase activity by doxorubicin was totally prevented by preincubation with nicorandil. Nicorandil also concentration-dependently (10 to 100 μM) decreased doxorubicin-induced ROS and the effect was antagonized by 5-hydroxydecanoate. The inhibition profile of doxorubicin-induced ROS by nicorandil was unaltered when an L-arginine derivative or a protein kinase G inhibitor was present. Preincubation with pinacidil mimicked the effect of nicorandil and the protection was eliminated by glibenclamide. Quantitative colocalization of fluorescence indicated that the mitochondrion was the target organelle of nicorandil and the observed response was a decrease in the mitochondrial inner membrane potential. Interference with H+ movement across the mitochondrial inner membrane, leading to depolarization, also protected from doxorubicin-induced ROS. The data indicate that activation of the mitochondrial ATP-sensitive K+ channel by nicorandil causing mitochondrial depolarization, without participation of the NO donor activity, was responsible for inhibition of the mitochondrial NADPH oxidase that is the main contributor to ROS production in cardiomyocytes. Impairment of the cytosolic Ca2+ signal induced by caffeine and the increase in lipid peroxidation, both of which are indicators of doxorubicin-induced oxidative stress, were also prevented by nicorandil.The primary cardiotoxic action of doxorubicin when used as antitumor drug is attributed to the generation of reactive oxygen species (ROS) therefore effective cardioprotection therapies are needed. In this sense, the antianginal drug nicorandil has been shown to be effective in cardioprotection from ischemic conditions but the underlying molecular mechanism to cope with doxorubicin-induced ROS is unclear. Our  Doxorubicin-induced ROS overproduction occurs inside mitochondria and is mediated by the mitochondrial NADPH oxidase (mitoNOX) activity   + effluxe in ΔΨm  offered ATP opening without the participation of the NO/cGMP-dependent pathway. It is suggested that mitoKATP activation by nicorandil, giving rise to mitochondrial depolarization, is responsible for mitoNOX inhibition and the subsequent lack of ROS production. Modulation of ΔΨm may be a promising therapeutic strategy for the treatment or prevention of cardiac disorders related with ROS overproduction.In conclusion, although nicorandil exhibits a dual pharmacological action, the cardioprotective effect was exerted by mitoK


The pre-TIPS study in 1994–95 showed that the duration of untreated psychosis (DUP) was long in our region with a mean value of 2.1 years, and median 26 weeks. This set the stage for the TIPS-study (1997–2000), reducing DUP through information campaigns targeted to the general population and other referral agents  in Rogaland County (Norway). The information campaigns were launched together with a low threshold organization with direct access to an early detection team, for a diagnostic interview and help. The hypothesis was that this could change help seeking behavior, awareness towards psychosis and thus reduce the DUP. The information campaigns and the early detection team were introduced in an early detection(ED) area  comparing DUP with two usual-detection control sites in Oslo (Norway) and Roskilde (Denmark). As a result, DUP in the early detection area was reduced from 26 weeks median to 4 weeks median. Symptom and function advantages of early detection and DUP reduction have been demonstrated as being significant throughout the follow-up period. Social and functional outcome have been increasingly emphasized as being key parameters, as these contribute to both quality of life and to financial costs in society. The TIPS program continues to include and follow patients; since the mid 2000’s also young people with ultra-high risk states  or substance induced psychoses.Previous ultra-high risk studies suggest that psychotic disorders are associated with structural and functional abnormalities within the frontoparietal circuits and that medication status is a potential confounding factor. We investigated neural correlates to ultra-high risk (UHR) for psychosis in medication-naïve patients.41 CHR patients and 37 healthy controls were examined with 1.5 Tesla MRI, yielding functional scans while performing an N-back task and structural T1-weighted brain images. Functional and structural data underwent automated preprocessing steps in SPM and Freesurfer, correspondingly. The groups were compared employing mass-univariate strategy within the generalized linear modelling framework.UHR demonstrated reduced suppression of the medial temporal lobe (MTL) regions during n-back task. We also found that, consistent with previous findings, UHR subjects demonstrated thinning in prefrontal, cingulate, insular and inferior temporal areas, as well as reduced hippocampal volumes.The present findings add to the growing evidence of specific structural and functional abnormalities in the brain as potential neuroimaging markers of psychosis vulnerability.


The neural control of social behaviors in rodents requires the encoding of pheromonal cues by the vomeronasal system. Here we show that the typical preference of male mice for females is eliminated in mutants lacking oxytocin, a neuropeptide modulating social behaviors in many species. Ablation of the oxytocin receptor in aromatase-expressing neurons of the medial amygdala (MeA) fully recapitulates the elimination of female preference in males. Further, single-unit recording in the MeA uncovered significant changes in the sensory representation of conspecific cues in the absence of oxytocin signaling. Finally, acute manipulation of oxytocin signaling in adults is sufficient to alter social interaction preferences in males as well as responses of MeA neurons to chemosensory cues. These results uncover the critical role of oxytocin signaling in a molecularly defined neuronal population in order to modulate the behavioral and physiological responses of male mice to females on a moment-to-moment basis. Oxytocin is a hormone that promotes milk production, contractions during childbirth, and many social interactions in humans and other creatures. It has also been implicated in conditions like autism or schizophrenia, which show altered social interactions. Oxytocin is made and released by cells in the brain called neurons. The oxytocin-producing neurons are clustered in a brain region called the hypothalamus, and oxytocin can act over a long distance in the brain or in the body. Many mammals detect chemical signals called pheromones that are involved in social interactions. These chemicals are detected by neurons in a structure within the cartilage of the nose called the vomeronasal organ. Pheromone-sensing neurons in the vomeronasal organ connect with another part of the brain called the medial amygdala. The medial amygdala, in turn, connects with regions of the brain that control behavior.Mice in particular rely on pheromones for social communication. Male and female mice respond differently to pheromones. Male mice prefer to investigate female mice to other males. The neurons in medial amygdala of male mice also become more active in response to scents from females than from males. Oxytocin is known to act on the medial amygdala, but its exact role in the male’s preference for females and their scents is not known.Now, Yao et al. show that oxytocin controls male preference for interacting with females and their scents by turning on neurons in the medial amygdala. In the experiments, male mice genetically engineered to lack oxytocin do not prefer female mice to other males, and they also appear unable to distinguish male and female scents. These mice also have less activity in the neurons of the medial amygdala when exposed to females and their scents. Directly manipulating these neurons and the oxytocin receptors on them also altered sex-preferences in male mice.The experiments show that oxytocin alters the behaviors of male mice in response to females or their scents by manipulating a specific set of brain cells. More studies of these cells or their interactions with oxytocin might help scientists understand oxytocin-liked diseases that impair social interactions or develop new treatments for conditions like autism or schizophrenia. A fundamental goal of neuroscience is to understand how brain circuits control behavior. New advances in genetic, imaging and functional approaches applied to small and large brains are uncovering wiring diagrams and neural activity ensembles underlying specific behaviors at increasingly high resolution. Proper regulation of the functional properties of these neural circuits requires cohorts of still poorly understood neuromodulators, such as biogenic amines and neuropeptides . ReleaseThe vertebrate hypothalamus has emerged as a particularly rich source of neuropeptides. These neuromodulators shape intricate allostatic and behavioral functions such as temperature and energy balance, thirst, hunger, sleep, as well as aggression, reproduction, and parenting. In particular, the nonapetides oxytocin (OXT) and vasopressin (AVP) represent an important set of neuromodulators that are produced by discrete populations of hypothalamic neurons in response to social signals and according to an animal's physiological state . These nGenetic models of mice deficient in OXT or in OXT receptor (OXTR) have helped uncover the requirement of OXT signaling in myriad social behaviors including recognition of familiar and novel conspecifics , parentaIn mice, genetic and surgical ablation of the vomeronasal organ (VNO), a specialized organ required for the detection of pheromones, results in atypical behaviors towards conspecifics, with female and male mutants displaying sexual behaviors indiscriminately towards conspecifics of both sexes, suggesting that the vomeronasal system plays a critical role in decoding sex-specific chemosensory cues . The medHere, we investigated OXT action on neural circuits regulating sex-specific behavioral responses to conspecific signals. We found that, during social interactions, OXT mutant males do not display the preference for female conspecifics that is typical of wild-type males, and are impaired in chemosensory discrimination of sex-specific cues. To dissect the underlying cellular components of OXT function in behavioral sex-discrimination, we conducted genetic ablation as well as virus-mediated manipulation of OXTR, and demonstrate the critical role of OXT signaling in a sub-population of MeA neurons expressing the steroid converting enzyme aromatase. We further show that impaired chemosensory discrimination in OXT mutants correlates with altered chemosensory response profiles in the MeA. Moreover, acute modulation of OXT signaling in adults is sufficient to alter sensory representation in the MeA and sex discrimination in social interactions. Our study thus reveals that acute neuromodulatory function of OXT in a single, molecularly defined, population of neurons shapes the sensory representation of the MeA and is critical for tuning an animal's preference for male versus female social cues.-/-Oxt) and wild-type littermate controls (+/+Oxt) in investigating female versus male conspecifics  resulting in a positive social preference score that is significantly higher than one obtained with empty wire cups  and had a social preference score that was not significantly different from one obtained with empty wire cups . Thus, our results suggest that loss of OXT alters the normal preference of male mice to interact with female rather than male conspecifics.The amount of time subjects spent adjacent to each enclosure cage was quantified, and a 'social preference score'  was calculated that ranges from −1.0  to 1.0 . Data show that control +/+Oxt and -/-Oxt male littermates were presented in their home cages with male and female bedding separated by a divider. Subject mice were allowed to investigate the two types of bedding for a total of 5 min. As expected, +/+Oxt males spent a significantly longer time on average investigating female than male bedding , whereas -/-Oxt male mice presented lost preference in investigating female chemostimuli  was significantly higher than that of -/-Oxt male mice  as well as -/-Oxt   mice spent significantly less time in the goal zone containing fox urine in additional to a food pellet  and the AVP receptors V1aR, V1bR and V2R . To inveuse line , which euse line . Unfortuuse line , preventcontrol = 10, nmutant = 6; V1bR: ncontrol = 10, nmutant = 11; V1aR V1bR: ncontrol = 11, nmutant = 11), such that both control and mutants spent more time investigating female beddings than male beddings  and had positive mean odor preference scores  (http://mouse.brain-map.org/gene/show/21801); while aromatase, which converts circulating testosterone to estrogen, is mainly found in neurons of the posterodorsal subdivision of the MeA (MeApd)  . TRH is  (MeApd) .Trh-IRES-Cre knock-in line (Cyp19a1(aromatase)-Cre BAC transgenic line that faithfully recapitulates the endogenous aromatase expression in the MeApd, as well as in the other described sites of endogenous aromatase expression across the brain, such as the posteromedial BNST, preoptic hypothalamus, and lateral septum  or TRH . In all cases, male mice with intact OXT signaling were used as controls for littermates lacking the expression of OXTR in specific neuron types. Data show that male mice lacking OXTR expression in aromatase-positive neurons failed to show a significant preference for social interactions with female conspecifics, in a manner similar to what we observed previously with -/-Oxt mice  and male mice with impaired OXT signaling restricted to TRH neurons  all spent significantly longer durations in the female interaction zone and had a strong preference for female conspecifics  and Cyp19a1-Cre  male mice, both of which have intact OXT signaling. In contrast, the homozygous offspring of these two lines, which have impaired OXT signaling in aromatase-positive neurons, showed loss of preference to investigate female bedding  or TRH positive neurons  still spent significantly longer durations in the goal chamber with the attractive cue of either food or female urine than the goal chamber containing fox urine , another first order target of neurons in the AOB, and a key region critical for the processing the chemosensory information  in the bilateral BNST or MeA , whereas control  and male mice with impaired OXT signaling in the BNST  presented normal preference in investigating female bedding , whereas impaired OXT signaling in the BNST had no effect on the odor preference . Thus, our results suggest that aromatase-positive neurons of the MeA, but not of the BNST, mediate the effect of OXT on the discrimination of sex-specific cues by male mice.Taken together, our results demonstrate that aromatase-positive neurons represent a critical site for OXT action in the control of chemosensory discrimination of male versus female conspecifics. Yet, the ablation of OXTR from aromatase-positive neurons using the ormation . To deteT or MeA . Mice re bedding . The odoflox/floxCyp19a1-Cre; Oxtr male mice. A recombinant AAV virus in which the CMV promoter drives constitutive expression of OXTR (AAV-OXTR) or a control virus with CMV-driven expression of GFP was stereotaxically delivered . Identification of single unit activity was based on spike shape, clustering of principal component projections, and a clear refractory period between successive spikes . Consistflox/floxOxtr male mice were compared to that of mutant male mice lacking OXTR signaling in aromatase-positive neurons. A total of 106 single units were recorded from the MeA of control male mice , with 40 units responding to VNO cues with a p value <= 0.01 (non-parametric ANOVA) and 191 single units were recorded from the MeA of mutant male mice , with 70 units responding to VNO cues with a p value <= 0.01 (non-parametric ANOVA).Sensory-evoked responses were identified by comparing the spike rates before stimulus presentation (20 s prior to stimulus presentation) to spike rates following stimulus presentation (40 s following stimulus presentation) using a non-parametric ANOVA. Units were considered 'responsive' if any stimulus elicited a p-value less than 0.01. MeA neurons from control Single units responsive to VNO cues were categorized by the sensory stimulus that elicited the strongest response: female, male or predator . In acco−1  at a dosage of 5 mg/kg−1 , followe−1  and elec−1 . We foun−1 , without−1 . The chaflox/floxOxtr male mice were maintained for the entire experiment, which typically lasted 2 hr .To assess the acute function of OXT signaling at the sensory response level, we compared the responses of single MeA neurons before and after systemic OXTR antagonist injection . Before Data show that OXTR antagonist reduced the number of neuronal responses categorized as 'female' . OXTR antagonist application dramatically reduced the strength of female-responsive units  and the supraoptic nucleus (SON). To systematically map the distribution and origins of OXT fibers from the two populations of OXT-expressing neurons, a Cre-dependent AAV-ChR2-YFP virus that readily enables visualization of neuronal fibers was stereotaxically delivered to either the PVN or the SON of an OXT-iCre line . Cre-dependent AAV virus conditionally expressing the inhibitory DREADD (AAV8-hSyn-DIO-hM4Di-mCherry) was stereotaxically delivered either to the PVN  or the SCre line   to allow the activation of PVN OXT-expressing neurons by IP injection of CNO . These results are consistent with a relatively short duration (~15 min) of CNO driven enhanced OXT activity, followed by a long-lasting inhibition of OXT signaling, which may result from depletion of presynaptic OXT and/or desensitization of MeA OXTR.In mice, social preference for male and female conspecifics has been shown to emerge during puberty, resulting in male mice preferentially investigating female stimuli over male stimuli . Here, wAVP and OXT regulate overlapping social behaviors with the specific function of the two peptides differing according to the sex and age of the animal, and the target neuronal population . For exaOur results reveal a robust specificity in the role played by OXT in regulating the sensory processing of sex-specific social cues in males. We extend previous findings by showing that in the 3-chamber-social-investigation paradigm OXT, but not AVP, plays a unique role in the discrimination of sex-specific cues by males and that this signaling is exclusively mediated by OXTR in the MeA, and does not involve V1aR nor V1bR. Moreover, despite the well-established cross reactivity of OXT and AVP on all of OXTR, V1aR and V1bR, and the presence of rich AVP fiber tracks in the MeA, it appears that endogenous AVP does not compensate the defects in social interactions resulting from loss of OXT.Our behavioral experiments revealed little bias in the time female mice spent interacting with male versus female mice. We note that this result differs from previous reports in which a preference of intact female mice for male-derived chemo-stimuli or intact males was demonstrated . This diOXT has been implicated in the modulation of a large number of social behaviors, including social recognition and social learning , the forflox/floxCyp19a1-Cre;Oxtr male mice, whereas expression of OXTR in the BNST alone has no effect, thus helping to further delineate the site of OXT action to aromatase-expressing neurons of the MeA.To further investigate mechanisms underlying the role of OXT in male mouse social preference, we used genetic and viral tools to conditionally impair OXT signaling in distinct brain regions and neuronal populations, and mapped the site of OXT action to the sexually dimorphic aromatase-expressing neurons of the MeA. Furthermore, virus-mediated expression of OXTR in the MeA rescues sex-specific odor discrimination impairment in This in turn led us to perform in vivo multisite extracellular recordings and directly monitor the responses of individual MeA neurons to conspecific and predator stimuli with and without OXT signaling. Our results showed that, in the absence of OXTR, MeA neurons display overall reduced responses to chemosensory cues, while the percentage of neurons responding to male cues is increased, suggesting that the lack of preference of OXT mutant mice for sex-specific cues results from abnormal patterns of MeA neural activity. Moreover, consistent with a specific role of OXT in the processing of conspecific cues, neuronal responses to mouse cues are reduced more than those to predator cues in OXT mutants. Importantly, the observed behavioral and neural phenotype observed in OXT mutants may result from defects in early OXT-dependent developmental processes, or from the acute lack of OXT-mediated signaling. Our data showing that similar sex-preference behavioral defects are observed in constitutive as well as in conditionally virus-mediated OXTR ablation in the adult MeA strongly suggest a role for OXT signaling in adult. This conclusion is further supported by the ability of OXTR-expressing viral injection in the adult MeA, but not BNST, to rescue the phenotype of OXTR mutants, and by the observation of behavioral and neural phenotypes resulting from acute reduction of OXT activity in adults using chemogenetic or pharmacological approaches. Therefore, the effect of OXT mediated through OXTR in aromatase-expressing neurons is not developmental, but rather acts on a moment-to-moment basis even in adults.Our previous study showed that the establishment of the sexually dimorphic sensory representation in the MeA requires steroid signaling near the time of puberty to organize the functional representation of sensory stimuli . In contRecent studies have shown that specific activation of aromatase-expressing neurons in the MeA promotes aggressive behaviors , and thaThe change in sensory responses observed in the MeA parallels the changes observed in male social preference, suggesting that the observed behavioral effect of OXT signaling is achieved by filtering which sensory responses have access to behavioral centers in the hypothalamus on a moment-to-moment basis. We showed that OXT enhances the responses of neurons in the MeA that encode female stimuli while suppressing sensory responses to both male and predator stimuli. This increased salience of female stimuli observed in this study is reminiscent of the strengthened responses for attended auditory and visual stimuli . OXT is An intriguing question raised by our data is how is the specificity of OXT action to conspecific responses being achieved in the MeA. Our study identifies aromatase-expressing neurons in the MeA as a necessary and sufficient site for OXT to shape the social preference of male mice to explore female stimuli. OXT has been shown to regulate specific brain functions by facilitating the release of other neurotransmitters, in particular dopamine and serotonin . In addiAltogether, our results uncover aromatase-expressing MeA neurons as a hub for OXT-dependent modulation of female-evoked neural responses and behaviors in the male mouse. OXT selectively increases MeA responses to female stimuli, indicating that OXT signaling has the ability to differentially modulate the specific sensing of female cues through its action on MeA neurons co-expressing aromatase and OXTR. The MeA receives direct sensory input from the AOB and is reciprocally connected with hypothalamic behavior centers. Although sex-specific sensory cues are relayed separately at the receptor level, AOB mitral cells, which send input to MeA neurons, often respond to multiple sensory stimuli. It is possible that MeA neurons co-expressing aromatase and OXTR receive input from AOB neurons that are preferentially responsive to female stimuli. Alternatively, the specificity of OXT in modulating female-evoked neural responses may be achieved by regulating the local MeA circuitry, for example by acting on a specific set of local aromatase- and OXTR-expressing interneurons that boost responses to female cues while dampening responses to male and predator signals. These possibilities are not mutually exclusive. Understanding the molecular identity and circuit connectivity of neurons both directly and indirectly modulated by OXT will be essential for understanding how OXT regulates female-evoked neural responses and behaviors in male mice.The data presented here provide significant new insights into the role of OXT signaling in the control of social behavior. OXT selectively enhanced neural and behavioral responses to female stimuli, suggesting that subsets of female-responsive and male-responsive neurons in the MeA likely express different levels of OXTR. Thus, by dynamically and selectively tuning the neural representation of social cues in a genetically defined neuronal population of the MeA, OXT may alter the behavioral significance of social stimuli on a moment-to-moment basis.ad libitum. Animal care and experiments were carried out in accordance with the NIH guidelines and approved by the Harvard University Institutional Animal Care and Use Committee.All mice were maintained in 12 hr:12 hr light:dark cycles with food and water available tm1.1WsyOxtr/J, stock number: 008471) were from Jackson laboratories. The line had been backcrossed to the C57BL/6J inbred mice for at least 11 generations. V1aR knockout mice  and V1bR knockout mice  were obtained from Jackson laboratories and were backcrossed to the C57BL/6J inbred mice for at least six generations in our facility. The Oxt-iCre and Trh-Cre transgenic mouse lines were previously reported -reported . The linCyp19a1-Cre line was generated by BAC recombination, such that the Cre coding sequence was inserted in front of the start codon of the Cyp19a1 gene, which encodes aromatase. The ATG codon of the Cyp19a1 gene in the BAC was additionally mutated into a TTG stop codon.The The absence of germline recombination events due to the presence of Cre and floxed alleles together in the germline was confirmed by two genotyping strategies. One pair of genotype primers  was designed to amplify both WT and floxed loci of the conditional OXTR knockout allele . Another pair of primers  was designed to amplify the recombined locus (485 bp). The germline Cre-mediated deletion of floxed loci can be detected due to the lack of PCR products corresponding to the floxed loci (341 bp), and the presence of PCR products corresponding to the recombined locus (485 bp). We did not observe any germline recombination in our crosses.Cyp19a1-Cre line by generating a double transgenic line, consisting of the Cyp19a1-Cre line and the Rosa26-lsl-tdTomato reporter. Mice containing both Cyp19a1-Cre and Rosa26-lsl-tdTomato were bred to maintain the double-transgenic line. The expression of tdTomato was observed for multiple generations: no germline-mediated recombination was observed in this double-transgenic line, and the tdTomato expression pattern faithfully reflected the endogenous expression pattern of the aromatase gene, thus excluding the possibility of germline recombination or partial recombination.The absence of germline recombination and partial recombination due to activity in the early embryo was additionally confirmed for the The AAV-GFP and AAV-GFP-Cre viruses of serotype one were ordered from Penn Vector Core; the AAV-hSyn-DIO-hM4Di-mCherry virus and AAV-hSyn-DIO-hM3Dq-mCherry of serotype eight were obtained from the University of North Carolina Vector Core. To generate AAV-OXTR-IRES-GFP virus, the CMV-OXTR-IRES-GFP-BGH PolyA cassette was inserted into the XbaI site of pSub801, and the resulting plasmid was used to generate high titer virus of serotype one in the Harvard virus Core.Mice 6–8 weeks old were anesthetized by ketamine/xylazine. A total of 200 nl virus was stereotaxically injected into the target areas. The coordinates used for the PVN injections were: bregma: anterior-posterior, –0.90 mm; dorsal-ventral, –4.75 mm; lateral,±0.25 mm, those for the SON were: bregma: anterior-posterior, –0.80 mm; dorsal-ventral, –5.50 mm; lateral,±1.3 mm, those for the posterior medial amygdala were: bregma: anterior-posterior, –1.90 mm; dorsal-ventral, –5.0 mm; lateral,±1.75 mm, and those for the BNST were: bregma: anterior-posterior, –0.34 mm; dorsal-ventral, –4.50 mm and −4.00 mm; lateral, ±0.75 mm. Mice recovered for 4 weeks after surgery before being individually caged for social behavior tests.Mice brains were dissected and fixed overnight in 4% paraformaldehyde at 4°C, and 50 μm sections were prepared using a vibratome. Brain sections were blocked in PBX (PBS with 0.05% Triton X-100) containing 10% FBS for 1 hr at room temperature, following by overnight incubation in the appropriate primary antibodies at 4°C. The sections were then washed with PBX, and incubated with either Fluorophore-conjugated secondary antibodies or Biotin-SP-labeled secondary antibodies for 1 hr at room temperature. The sections were then incubated with fluorophore-labeled streptavidin when necessary, and mounted after washing in PBX.The following primary antibodies were used: rabbit anti-oxytocin , rabbit anti-vasopressin , and rabbit anti-GFP . Biotin-SP-AffiniPure Goat Anti-Rabbit IgG (111-065-144), and CY3 streptavidin (016-160-084) were from Jackson ImmunoResearch Laboratories, West Grove, PA. Alexa Fluo 488 Anti-mouse IgG, Alexa Fluo 488 streptavidin was from Invitrogen, Waltham, MA, .−1 acetic anhydride) for 10 min, and then incubated with the pre-hybridization solution  for 2 hr. The slides were then added with the hybridization buffer containing the appropriate DIG or FITC labeled probes and subsequent hybridization was carried out in a sealed chamber at 68°C overnight. The brain sections were then washed sequentially in 5XSSC, 0.2XSSC and TNT buffer . After the post-hybridization washes, slides were incubated with anti-FITC-POD  and/or anti-DIG-POD , followed by TSA amplification .RNA in situ hybridization was performed as described previously . BrieflyA stock solution was made by dissolving 5 mg of CNO  in 1.333 ml dH2O to make 3.75 mg/ml solution (10 mM). A working solution was prepared by diluting the working solution with normal saline at a ratio of 1:100. Mice received IP injection of CNO at a dose of 0.3 mg/kg, followed by behavioral analysis. To observe acute behavioral effects, mice were injected with CNO once, followed by behavioral test one hour later.−1, followed by behavioral analysis and electrophysiological recording.The OXTR antagonist  was purchased from Tocris Bioscience, Bristol, United Kingdom. A 10 mg/ml stock solution of OXTR-A was prepared in dH2O, and was delivered by intraperitoneal injection at a dosage of 5 mg/kgAll behavioral tests were performed in the dark phase of the light/dark cycle. Mice aged between 2 to 4 months were used for behavioral tests.Female or male beddings were collected from group housed C57BL6 mice on the same day of the odor preference test and the beddings of the same sex cages were not changed for 72 hr. Subject mice were individually caged for one week before behavioral test. About 50 ml of beddings were taken using 50 ml conical tubes and used for each odor preference assay. Female and male beddings were presented simultaneously to the resident mouse in its home cage. The two piles of bedding were separated by a plastic divider. The subject mouse was allowed to investigate the bedding for 5 min and the entire duration of the test was videotaped. The time spent by the test mouse to investigate each pile of beddings was quantified using the Observer software . The odor preference score was calculated as /.Urine was collected from five adult group-housed C57BL/6 males and five adult group-housed C57BL/6 female. Urine samples from the same sex were pooled and stored in at −80°C. Before testing each day, aliquots of urine were thawed and used. Subject mice were housed alone for a minimum of 3 days before the odor habituation/dishabituation test. The subject mice first received three 2 min presentations of saline at 1 min intervals, followed by three 2 min presentations of one urinary odor and finally by three 2 min presentations of a second urinary odor. Fisher’s LSD test was used to compare the difference between the durations that resident spent investigating the stimuli.A 111.8 cmx111.8 cm plexiglass arena was divided into 3 chambers of equal size. The left and right chambers each contained a flipped pencil holder (enclosure cage) with a 200 ml glass beaker on top. To test social preference, the two doors leading to the side chambers remained closed initially and the male mouse was allowed to acclimate to the central chamber for 10 min. In the habituation stage of the assay, the pencil holders in the side chambers were empty and the mouse was allowed to explore the side chambers for 10 min. In the social interaction phase, an unfamiliar C57BL6 mouse was placed in one of the cages and a second C57BL6 mouse of opposite sex was placed in the other cage. The subject mouse was then allowed to explore the arena for 10 min. The motion of the subject mouse during the habituation and social interaction phases of the assay was automatically tracked by Ethovision (Noldus). The time spent by the mouse in each chamber and in each interaction zone and the total distance travelled were calculated automatically by Ethovision. The interaction zone was defined as the area within 5 cm from the border of the enclosure cage. The social interaction preference score was calculated as /.To observe the acute effect of OXTR-A on social preference of male mice, subject mice were first tested in the 3-chamber social preference paradigm after saline injection, and then the preference was tested again 30 min after mice received IP injection of OXTR-A.flox/floxCyp19a1-Cre; Oxtr, a clean food pellet or one spiked with 25 μl of fox urine  served as the odor stimuli pair and were placed on the petri dishes in the left and right goal zones. For experiments involving flox/floxTrh-Cre; Oxtr, 20 μl of female mouse urine or fox urine was added to a cotton pad and then placed on the petri dish. The subject mouse was allowed to explore the Y-maze for 10 min. The motion of the subject mouse during the habituation and test phases of the assay was tracked and the duration spent by the mouse in each goal zone was calculated automatically by Ethovision.The subject mouse was allowed to acclimate to the starter chamber for 10 min. In the habituation stage of the assay, the left and right goal zones contained only an empty 10 cm petri dish and the mouse was allowed to explore Y-maze for 10 min. In the test phase, odor stimulus pairs were added to the petri dishes in either the left or right goal zones. For experiments involving OT mutants and All behavioral comparisons were analyzed using the Prism6 software to determine the statistical significance of behavioral results. Statistical tests for odor and social preference were performed by comparing the bias in time an animal spent near stimulus A  versus stimulus B  to the bias in time spent in the same physical area without the respective stimuli (ex. empty cups). In all cases, a family-wise error rate correction for multiple comparisons was made using the Holm-Sidak's method. Thus, our statistical tests account for any observed bias in an animal's preference for one stimulus while accounting for any innate spatial biases the animal may display.2 craniotomy was opened dorsal to the MeA based on stereotaxic coordinates, to allow the insertion of Neuronexus probes  coated with a fluorescent dye  into the MeA . All sensory stimuli were presented at least six times in a randomized order for each dataset. VNO stimuli were applied by placing 1 ul of stimulus (1:100 dilution in Ringer's) directly into the nostril, followed by activation of the sympathetic nerve cuff electrode to facilitate VNO pumping and stimulus entry to the VNO lumen. The nasal cavity and VNO was cleaned with Ringer's solution after each stimulus presentation .Animals were maintained under anesthesia while CNO or the OXTR antagonist were injected , and the electrophysiological recording was maintained throughout to allow identification of electrode movement due to the injection. Only units that were maintained throughout the recording were analyzed.https://github.com/joebergan/Spike-sorting) were used to extract 3.5 ms spike waveforms from the continuous data  and digitized at 25 kHz using an RZ2 processor, PZ2 preamplifier, and two RA16CH head-stage amplifiers . Custom MATLAB  scripts , and autocorrelation functions . Single units displayed a distinct spike shape that was fully separated from both the origin (noise) and other clusters (multi-unit) with respect to at least one principal component projection, and also displayed a clear refractory period in the interspike interval histogram.Valid sensory responses were identified by comparing the spike rate after sensory stimulation to the spike rate immediately preceding sensory stimulation for each unit. Responsive units passed a non-parametric ANOVA statistical test performed at the significance level of p≤0.01. Response magnitude was quantified as the change in average firing rate during the 40 s following stimulus presentation relative to the firing rate during the 20 s prior to stimulus presentation. Unless otherwise noted, statistics comparing two populations of units were performed using a nonparametric permutation test  as obser In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been favorably evaluated by Huda Zoghbi (Senior Editor) and three reviewers, one of whom, Richard D Palmiter (Reviewer #1), is a member of our Board of Reviewing Editors. The following individual involved in review of your submission has agreed to reveal their identity: W. Scott Young (Reviewer #3).Thank you for submitting your article "Oxytocin Signaling in the Medial Amygdala is required for Sex Discrimination of Social Cues" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:This paper provides important advances in understanding how oxytocin affects social behavior by identifying post-synaptic, aromatase/oxytocin receptor neurons in the medial amygdala that regulate perception of sex-related odors. Before final acceptance, a few concerns need to be resolved.Essential revisions:1) Behavioral alterations are mapped to circuit alterations but without clear causal explanations for how these behavioral effects are mediated. The finding that OXT specifically alters the responses to female stimuli are interesting, but the claim that the effects of acute OXTR blockade are surprising is not well supported. The electrophysiological analysis of the responses of MeA neurons provides some insight but questions about why the alteration of responses is sex-specific receive little attention and I believe that these are among the most interesting findings of the manuscript.2) Authors should emphasize the controls that were used when using DREADD and CNO to rule out non-specific effects of CNO.3) The possibility that genetic background might influence the role of vasopressin receptors should be addressed.4) The authors should do social preference tests after the intense grooming subsides when using the excitatory DREADD as suggested.5) The authors should discuss their results in broader context, if possible. What is the overall impact of poor social recognition by males? Does this lack of preferential recognition of females affect mating behavior, fertility or post-partum behaviors of males?6) Authors should indicate what experiments they did to be sure that all of the mice used for experiments did not have unexpected recombination events due to having Cre and floxed alleles together in the germline. Essential revisions:1) Behavioral alterations are mapped to circuit alterations but without clear causal explanations for how these behavioral effects are mediated. The finding that OXT specifically alters the responses to female stimuli are interesting, but the claim that the effects of acute OXTR blockade are surprising is not well supported. The electrophysiological analysis of the responses of MeA neurons provides some insight but questions about why the alteration of responses is sex-specific receive little attention and I believe that these are among the most interesting findings of the manuscript.We agree with these points and have modified the text accordingly:- In absence of slice recording, which is well beyond the scope of the present study, any mechanistic scenario remains hypothetical, but we agree that a set of plausible mechanistic hypotheses for how the observed behavioral effects are mediated is needed. We have added specific text in the Discussion that details possible circuit-level scenarios .- The dynamic regulation of OXT by external environment and internal physiological state is well-recognized , and the ability of acute OXTR blockade to modulate behaviors has also been reported . In our initial submission, our intention was to emphasize the acute effects of OXT in social preference in contrast to a potential role in development, and we did not mean to claim any novelty with respect to the acute effects of OT. We have now clarified the text accordingly.eLife 2014) has demonstrated that the establishment of the striking sexually dimorphic sensory representation in the MeA requires steroid signaling near the time of puberty to organize the functional representation of sensory stimuli. In the present work, we show that virally expressed OXTR restores social preference in the adult, indicating a role of OXT in dynamically regulating social preference in adults, in contrast to an organizational role. Following the reviewer’s suggestion, we have clarified in the text that acute behavioral effects of OXTR blockage have been reported previously, and have phrased the conclusions from our current work accordingly.Indeed, our past study  a sex-specificity in the sex of the tested animals, and (2) a sex-specificity in the response of the tested animals to cues from males versus females:1) We have focused on the function of OXT in male mice because they reliably demonstrate a strong social preference to explore female mice. By contrast, female mice of all tested strains reliably displayed a small to non-significant social preference for females in similar behavioral assays. This is made more explicit in the Results and Discussion sections.2) In male mice, the alteration of neuronal responses due to loss of OXT signaling differentially affects the responses to female versus male and predator cues. There are several potential mechanisms consistent with the observed results, which all involve the presence of distinct channels of sensory information for female stimuli, whose modulation by oxytocin occurs in aromatase-positive neurons and are separable from male- and predator-specific sensory sensing. We now describe candidate mechanisms more thoroughly in the Discussion.We thank the reviewers for these suggestions, which have helped further clarify the significance and broader context of our results.2) Authors should emphasize the controls that were used when using DREADD and CNO to rule out non-specific effects of CNO.In view of recent findings on the effects of CNO and CNO metabolites on brain function, it is indeed imperative to have appropriate controls for any DREADD experiment. In our study, we compared groups in which DREADDs were expressed in different brain areas. CNO only induced changes in social preference when paired with DREADD expression in the PVN, but not in the SON – confirming that the effect of CNO is specific to DREADD infected OXT neurons in the PVN. We now further clarify the concerns of the specificity and function of DREADDs in the text as follows:“Two sets of controls confirmed that the observed DREADD-mediated effects of CNO are specific to the activation or inhibition of OXT-expressing neurons. […] Second, the OXT-iCre male mice expressing the inhibitory DREADD in the SON presented normal preference towards female mice before and after CNO injection, excluding the possibility that the impaired social preference is due to an off-target effect of CNO or its metabolite clozapine .”3) The possibility that genetic background might influence the role of vasopressin receptors should be addressed.This is indeed an important point that we had failed to address in our initial submission. The OXT KO, Avpr1a and Avpr1b lines were from the Jackson Laboratory. All lines were backcrossed to C57BL/6 for >6 generations, in order to avoid the impact of different genetic background on social behaviors. We did not observe any impaired social preference of Avpr1a and Avpr1b knockout lines in our lines in the C57BL/6 background. This information has now been added to the text.4) The authors should do social preference tests after the intense grooming subsides when using the excitatory DREADD as suggested.We agree with the suggestion and have explored the behavioral effects of chemogenetic activation of OXT neurons after the initial grooming effects subside. The results are summarized below and were added to the manuscript.The excitatory DREADD virus, AAV8-DIO-hM3Dq-mCherry, was stereotaxically injected into the PVN of OXT-iCre male mice, and times spent investigating a pair of lightly anesthetized male and female stimulus mice  were meaAfter saline injection, OXT-iCre mice expressing excitatory DREADD in the PVN preferentially investigated the female stimulus mouse. By contrast, in experimental animals, central OXT levels were immediately enhanced after CNO injection, as indicated by lengthy hypergrooming observed post CNO injection. However, after the hypergrooming subsided (15 min after CNO injection) experimental animals showed a loss of female preference that did not recover even when tested 2 days, 1 week and 2 weeks after CNO injection .These data contrast with the results obtained by extracellular recordings, in which we observed a significant increase in the response strength of MeA neurons to female stimuli after CNO injection . InteresIn the electrophysiology experiments, a 5-minutes baseline recording was conducted post-injection, followed by 6 times of presentations of 4 stimuli, overall lasting around 25 to 30 minutes post-CNO injection . We foun5) The authors should discuss their results in broader context, if possible. What is the overall impact of poor social recognition by males? Does this lack of preferential recognition of females affect mating behavior, fertility or post-partum behaviors of males?We have extended the discussion on this topic. Briefly, we believe that OXT signaling provides an increase in the saliency of certain social stimuli in certain social contexts. Thus, the enhancement of sensory responses of male mice toward females is similar to the enhancement of visual responses observed in visual attention or the enhanced auditory responses observed in auditory attention.6) Authors should indicate what experiments they did to be sure that all of the mice used for experiments did not have unexpected recombination events due to having Cre and floxed alleles together in the germline.The authors should also attend to the minor, editorial comments.The absence of germline recombination was confirmed by two genotyping strategies: One pair of genotype primers are designed to amplify both WT and floxed loci of the conditional OXTR knockout allele . Another pair of primers are designed to amplify the recombined locus (485 bp). The germline Cre-mediated deletion of floxed loci can be detected due to the lack of PCR products corresponding to the floxed loci (341 bp), and the presence of PCR products corresponding to the recombined locus (485 bp). We did not observe any germline recombination in our crosses. This strategy can rule out germline recombination, but still cannot detect partial recombination due to early embryo activity. The above genotyping information has been added to the Materials and methods section.The absence of germline recombination and partial recombination due to activity in early embryo is additionally confirmed for the aromatase-Cre line by generating a double transgenic line, consisting of the aromatase-Cre line and the Rosa26-lsl-tdTomato reporter. Mice containing both aromatase-Cre and Rosa26-lsl-tdTomato were bred to maintain the double-transgenic line. The expression of tdTomato was observed for multiple generations. We did not observe any germline-mediated recombination in this double-transgenic line, and the tdTomato expression pattern faithfully reflects the endogenous expression pattern of the aromatase gene, thus excluding the possibility of germline recombination and of partial recombination due to activity in the early embryo.


PEGylation of DNA-encoded libraries enables high-throughput screening of small-molecule catalysts in organic solvents. in vitro selection for bond-formation using amphiphilic DNA-encoded small molecules charged with reaction substrate, which enables selections to be conducted in a variety of organic or aqueous solvents. Using the amine-catalysed aldol reaction as a catalytic model and high-throughput DNA sequencing as a selection read-out, we demonstrate the 1200-fold enrichment of a known aldol catalyst from a library of 16.7-million uncompetitive library members.We have developed a novel high-throughput screening platform for the discovery of small-molecules catalysts for bond-forming reactions. The method employs an  Such methods enable the screening of all library members simultaneously for catalytic activity, thus greatly increasing the throughput of catalyst discovery. One notable strategy for high-throughput combinatorial screening of small-molecule catalysts includes the one-bead-one-compound (OBOC) approach,1 which relies upon on-bead split-and-pool combinatorial synthesis of chemical libraries and subsequent on-bead or off-bead evaluation. This strategy has resulted in the recent discovery of catalysts capable of effecting chemo- and regioselective modifications on complex targets, including the site-selective epoxidation of polyenes,3 and reduction of polyunsaturated aldehydes.4 One of the major limitations of OBOC and other existing methods for catalyst discovery is the level of throughput. Without advanced infrastructure, multiwell and on-bead screening approaches generally limit the library size to less than 104, with most libraries typically being on the order of 103 or smaller. Approaches that can considerably increase the throughput of catalyst screening, while still enabling the power to multiplex the small molecule libraries for different reaction conditions and transformations, should greatly accelerate the discovery of small-molecule catalysts and structure–activity relationships.The en masse screening method is one that implements a selection pressure to remove catalytically inactive molecules from the library, thus leaving only the catalytically active species to identify. Selections offer significant advantages over traditional screening approaches including (i) a selection evaluates all molecules of a library simultaneously, regardless of library size8 and (ii) selections are typically easier to execute as they do not require the spatial separation of library members nor sophisticated equipment. In vitro selection has been a highly successful and powerful approach for the discovery of catalytic biopolymers from libraries containing greater than 1014 members.9 Several variants of in vitro selection for biopolymer catalysts exist, including phage display,10 mRNA display,11 ribosomal display,12 and DNA display.13 The overarching theme amongst these selection methods is that the biopolymer catalyst (phenotype) is spatially associated with its genetic code (genotype), thus PCR amplification and DNA sequencing can reveal the identity of those biocatalysts that have performed the desired catalytic function to survive the selection pressure. The in vitro selection of small-molecules from large DNA-encoded libraries14 has also been very successful, particularly in the discovery of small molecule drugs.17 While the application of this technology to medium-throughput screening of aqueous-tolerant reactions using hybridization-dependent microarrays has been successful,18 the application of this technology to the discovery of small-molecule catalysts using in vitro selection has remained unexplored. The primary reason for this is likely due to the poor solubility of DNA in non-aqueous solvents, where the majority of catalytic reactions operate. Herein, we demonstrate a new high-throughput selection platform for the discovery of small-molecule organic catalysts for intermolecular bond-forming reactions using DNA-encoded libraries in organic solvents.An alternative At the heart of the envisaged platform is a replicable amphiphilic DNA, which can be used to individually barcode members of a large combinatorial chemical library . The amp29 While most of these strategies involve the complexation of DNA with surfactants or the generation of nanogels, we were drawn to approaches that conjugated a single polymer to DNA to impart solubility in organic solvents. This would allow for the installation of this solubilising group distal to the site of catalysis, obviating any undesired interactions during catalyst selection. The conjugation of PEG 10 000 to ssDNA has been successful in enabling the solubility of DNA in a variety of organic solvents.24 This strategy has only been validated for short oligonucleotide sequences of up to 21 nt in length, which is too short for this selection system; therefore, we sought to determine if this approach could be extended to accommodate longer ssDNAs.The initial step toward realising this high-throughput catalyst-screening platform is the development of a modified DNA that is soluble in both water and commonly used organic solvents. Unmodified DNA is poorly soluble in anhydrous organic solvents, creating heterogeneous mixtures upon aggregation, limiting its use as an encoding element for catalyst selection in organic solvents. Several reports have described methods to increase the solubility of DNA in non-aqueous solvents.30 To determine the optimal polymer length for our system, we prepared a model 5′-amino modified 48 nt ssDNA sequence, which we conjugated to PEG-N-hydroxysuccinimide (PEG-NHS) esters ranging in average mass from PEG 10 000 to PEG 40 000. The influence of a PEG polymer on the solubility of ssDNA in organic solvents was determined by preparing 5 μM solutions of the PEGylated DNA in various solvents and analysing the samples by UV-Vis spectroscopy. Unfortunately, PEG 10 000 was unable to facilitate solubility of the 48 nt ssDNA into any solvents except water and methanol , which accommodates two 18 nt primer sites and a 12 nt catalyst-encoding region; a 12 nt region can encode greater than 16 million unique molecules by established split-and-pool tandem DNA/small-molecule synthesis methods.methanol . We begamethanol ; howevermethanol . ImportaR Fig. S2.32 and encouraged by the reported success of DNA-templated aldol reactions catalysed by proline-modified ssDNA in aqueous solvents,33 we implemented the secondary amine catalysed aldol reaction between a ketone and an aldehyde as our model. We designed a DNA architecture that would accommodate the catalyst site, the reactant site, and a PEGylation site, and could be readily synthesized by solid-phase DNA synthesis. We reasoned that the PEGylation site should be distal to the catalyst and reactant site to minimise interference on catalysis by the PEG chain. We also decided to incorporate a long spacer between the catalyst site and the reactant site to permit sufficient flexibility for the catalyst to comfortably engage the substrate. Thus, a 48 nt PEGylated ssDNA was synthesised to satisfy these specifications . Reaction success differentiates the product via biotin tag which, after incubation with streptavidin, allows visual comparison by a mobility shift between unreacted starting material and successfully catalysed reaction products using native gel electrophoresis. To determine the optimal reaction conditions, a solvent screen was performed for the aldol reaction in the various solvents previously concluded to efficiently solubilise the DNA-encoded catalyst architecture  DNA bases non-specifically react to form stable covalent adducts with the biotinylated aldol reactant; (ii) DNA catalyses the aldol reaction and results in non-specific biotinylation of DNA; (iii) the catalyst forms stable covalent adducts with the biotinylated aldol reactant; and (iv) inter-strand catalysis results in biotinylation of inactive library members by an active member. To address issues i–iii control experiments were designed to demonstrate that catalysis and bond-formation with biotinylated reactant happens only when the DNA molecule has both the catalyst and the aldol reactant attached . To addranalysis .The positive control was diluted 500-fold into a library of DNA sequences that lacked a catalyst. The library mixture was incubated with the biotinylated aldehyde reactant in DCE for three hours followed by binding to streptavidin-coated magnetic beads. After extensive washing of the beads, on-bead PCR was performed to amplify the selection survivors. Enrichment analysis was determined by restriction enzyme digestion of the PCR product, followed by non-denaturing PAGE analysis. After one round of selection for catalytic activity, the positive control was enriched 100-fold . Importa12) library members; however, instead of restriction digest as a readout, Illumina barcoded adapters were added to the template sequences by PCR amplification and Illumina Mi-Seq paired-end sequencing was performed (de novo discovery of small-molecule catalysts for bond-forming reactions.Satisfied with the outcome of the preliminary mock selections, high-throughput DNA sequencing was performed to quantitatively determine the fold enrichment of the aldol reaction selection under more dilute selection conditions. Compared to the EMSA characterisation, which only allows characterisation of one specific sequence, DNA sequencing permits characterisation of all the sequences in a library allowing for a more in-depth analysis of the selection outcome. Aldol selection was performed as described above with the positive control diluted 2000-fold into a library of 16.7 million (Nerformed . Post-seerformed  involvederformed . Sequencin-cis reactant and an in-trans biotinylated reactant. Affinity pull-down and readout by high-throughput DNA sequencing enables the rapid identification of active catalysts. Using the amine-catalysed aldol reaction as a model, we demonstrated that this approach can be implemented in various organic and aqueous solvents and can enrich a known aldol catalyst by 1200-fold. This platform has the potential to greatly accelerate the discovery of catalysts by increasing the throughput of catalyst screening efforts and expanding the chemical space explored during conventional catalyst screenings.In summary, we have developed a catalyst selection system based upon the use of DNA-encoded libraries in organic solvents. Survival of the selection requires DNA-encoded catalysts to engage in catalytic bond formation between an There are no conflicts to declare.


It was the primary purpose of our study to evaluate the inter- and intra-observer reliability of a standardized SPECT/CT algorithm for evaluating patients with painful primary total hip arthroplasty (THA). The secondary purpose was a comparison of semi-quantitative and 3D volumetric quantification method for assessment of bone tracer uptake (BTU) in those patients.A novel SPECT/CT localization scheme consisting of 14 femoral and 4 acetabular regions on standardized axial and coronal slices was introduced and evaluated in terms of inter- and intra-observer reliability in 37 consecutive patients with hip pain after THA. BTU for each anatomical region was assessed semi-quantitatively using a color-coded Likert type scale (0-10) and volumetrically quantified using a validated software. Two observers interpreted the SPECT/CT findings in all patients two times with six weeks interval between interpretations in random order. Semi-quantitative and quantitative measurements were compared in terms of reliability. In addition, the values were correlated using Pearson`s correlation. A factorial cluster analysis of BTU was performed to identify clinically relevant regions, which should be grouped and analysed together.The localization scheme showed high inter- and intra-observer reliabilities for all femoral and acetabular regions independent of the measurement method used (semiquantitative versus 3D volumetric quantitative measurements). A high to moderate correlation between both measurement methods was shown for the distal femur, the proximal femur and the acetabular cup. The factorial cluster analysis showed that the anatomical regions might be summarized into three distinct anatomical regions. These were the proximal femur, the distal femur and the acetabular cup region.The SPECT/CT algorithm for assessment of patients with pain after THA is highly reliable independent from the measurement method used. Three clinically relevant anatomical regions  were identified. Combined single photon emission computerized tomography and conventional CT (SPECT/CT) promises the combined assessment of anatomical and functional information and hence its value is increasingly recognized in orthopaedics –23.SPECT/CT has been reported to be beneficial in identifying the cause of patients`pain after total knee arthroplasty, patients with chondral or osteochondral lesions, before and after high tibial osteotomy and after ACL reconstruction –23. AlthDue to its specific characteristics SPECT/CT is more sensitive and specific than SPECT and CT alone. Clearly, it is the accurate anatomical localization of the SPECT-tracer uptake using the CT as reference map that promises improved diagnostic confidence, particularly in patients with pain after joint replacement surgery , 20, 23.Recently our group has described and validated a standardized diagnostic algorithm using SPECT/CT in patients with pain after total knee arthroplasty including analysis of bone tracer activity and position and alignment of the TKA , 23, 28.In addition, in clinical practice it is a pertinent question if quantification of BTU in SPECT/CT offers so much more information than formerly semi-quantitative methods to recommend its daily use. There is no current study comparing semi-quantitative and quantitative measurement of BTU in patients with pain after THA.Hence, it was the primary purpose of our study to evaluate the inter- and intra-observer reliability of a standardized SPECT/CT algorithm for evaluating patients with painful primary THA. The secondary purpose was a comparison of semi-quantitative and 3D volumetric quantification method for assessment of BTU in those patients.A consecutive series of 37 patients  presenting with hip pain after primary THA were prospectively collected and retrospectively included in this study. The data from all this patients were collected using our clinical information system . All patients had a primary THA with a maximum interval of 6 months from primary THA.The study was approved by the local ethical committee (EKNZ 205/10). Written informed consent was obtained from all patients.SPECT/CT was performed using a hybrid system  with a dual-head gamma camera and an integrated, 16x0.75-mm slice-thickness CT. All patients received a commercial 500–700 MBq Tc-99m-HDP injection . Scintigraphic images in anterior-posterior and lateral projection were taken in the perfusion phase (immediately after injection), the soft tissue phase (3–5 min after injection) and the delayed metabolic phase (2–3 h after injection). SPECT/CT was performed with a matrix size of 128x128, an angle step of 32, and a time per frame of 25 s.The CT protocol was modified according to the Imperial Knee Protocol, which is a low dose CT protocol that includes high-resolution 0.75 mm slices of the knee and 3 mm slices of the hip and ankle joints .The localization of bone tracer activity was recorded on a standardized localization scheme developed for use in patients after primary THA Fig. . This deEach femoral zone is represented with a number (1-14). The acetabulum (A) is divided into four zones .The highest activity grading on SPECT/CT for each area of the localization scheme was recorded semiquantitatively (0–10). In addition, it was noted whether the area of tracer activity extended to the bone prosthesis interface. In that case an additional “c” was added to the tracer activity value.In addition, BTU was also quantified in 3D using a voxel based measurement method. For BTU analysis  the 3D reconstructed datasets of the delayed SPECT/CT images were used. The anatomical areas represented by a previously validated localization scheme were 3D volumetrically measured in terms of SPECT/CT tracer uptake values  , 9. The Two observers interpreted the SPECT/CT findings in all patients two times with six weeks interval between interpretations in random order. Both were blinded to results from previous observations. The inter- and intra-observer reliability of the localization scheme and grading of the tracer activity was determined. Semi-quantitative and quantitative measurements were compared in terms of reliability. In addition, the values were correlated using Pearson`s correlation.Finally, a factorial cluster analysis of BTU was performed to identify clinically relevant regions, which should be grouped and analysed together.Data were analyzed using SPSS 16.0 . Sample size was calculated according to the reported estimates for reliability studies using intraclass correlation coefficients (ICCs) .p < 0.05 was considered statistically significant.The median differences in measurements between the two observers (inter-observer) and within the measurements of the first observer (intra-observer) were calculated. The intraclass correlation coefficients for inter- and intra-observer reliability were also calculated. ICC values range from 0 to 1. A value of 1 indicates perfect reliability, 0.81 to 1 very good reliability and 0.61-0.80 good reliability . For allThe localization scheme showed high inter- and intra-observer reliabilities for all femoral and acetabular regions independent of the measurement methods (semi-quantitative versus 3D volumetric quantitative measurements). Inter- and intra-observer reliability (intra class correlation- ICC) of 99mTc-HDP-SPECT/CT tracer activity using the localization and semi-quantitative BTU grading scheme for the acetabular and femoral zones  are presented in Table Inter- and intra-observer reliability (intra class correlation- ICC) of 99mTc-HDP-SPECT/CT tracer activity using the localization and 3D voxel based quantitative BTU grading scheme for the acetabular and femoral zones were for all regions neary perfect (ICCs > 0.90).The measured values of BTU activity in SPECT/CT for each anatomical region using the semi-quantitative versus 3D volumetric quantitative method are shown in Table The factorial cluster analysis showed that the anatomical regions might be summarized into three distinct anatomical regions Table The most important findings of the present study were twofold. Firstly, a high inter-observer and intra-observer reliability was found for grading and localization of the BTU activity independent of the investigated region. The localization scheme and BTU grading was reliable and easily applicable, which would make it understandable by most clinicians. A reliable localization and grading scheme is needed to standardize the evaluation of SPECT/CT data and make those comparable with each other. The Gruen classification is already widely used for assessment of periprosthetic radiolucencies, hence it was decided to adapt this scheme to the biomechanics of the hip reflecting bone remodeling and integration of the prosthetic hip components. It has also been used by others to report BTU findings in SPECT/CT , 32.In a recent pictorial review by Tam et al. dealing with THA the authors reported their SPECT/CT analysis and reporting system, which is in accordance with the one presented in terms of the localization scheme used . HoweverIn a retrospective study Jin et al. investigated the periprosthetic bone remodeling of THA using SPECT/CT . SPECT/CSecondly, a high correlation between both measurement methods was found for the distal femur. A moderate correlation was found for the proximal femur and the acetabular cup regions.The factorial cluster analysis showed that the anatomical regions might be summarized into three distinct anatomical regions These were the proximal femur, the distal femur and the acetabular cup region. In the study by Jin et al. the ROI analysis was done at five different locations  . The autHowever, Jin et al. questioned the need for routine quantification of BTU in patients with THA . Based oIn contrast, we believe that a better understanding of bone remodelling after THA reflected by typical BTU pattern distribution will help to improve the reporting and diagnosis when using SPECT/CT. However, until analysis of BTU activity could lead to a better diagnosis we need to achieve a more profound knowledge of normal and abnormal BTU distribution and activity in native and arthroplasty patients.Another limitation to gain wider acceptance for quantification methods among clinicians is the utility, availability and simplicity of these analysis methods. Clearly, these have to be robust, reliable and easy to perform.The study bears a few limitations to be considered. This is a well sized small pilot study aiming to evaluate the analysis algorithm in patients with THA undergoing SPECT/CT. The clinical value of the algorithm needs to be further evaluated in larger, homogenous cohorts. The standard deviations are high, which is due to inter-patient variability. A typical findings in metabolic imaging.The SPECT/CT algorithm for assessment of patients with pain after THA is highly reliable independent from the measurement method used. Three clinically relevant anatomical regions  were identified.


Many cancers have long been thought to primarily metabolize glucose for energy production—a phenomenon known as the Warburg Effect, after the classic studies of Otto Warburg in the early twentieth century. Yet cancer cells also utilize other substrates, such as amino acids and fatty acids, to produce raw materials for cellular maintenance and energetic currency to accomplish cellular tasks. The contribution of these substrates is increasingly appreciated in the context of glioma, the most common form of malignant brain tumor. Multiple catabolic pathways are used for energy production within glioma cells, and are linked in many ways to anabolic pathways supporting cellular function. For example: glycolysis both supports energy production and provides carbon skeletons for the synthesis of nucleic acids; meanwhile fatty acids are used both as energetic substrates and as raw materials for lipid membranes. Furthermore, bio-energetic pathways are connected to pro-oncogenic signaling within glioma cells. For example: AMPK signaling links catabolism with cell cycle progression; mTOR signaling contributes to metabolic flexibility and cancer cell survival; the electron transport chain produces ATP and reactive oxygen species (ROS) which act as signaling molecules; Hypoxia Inducible Factors (HIFs) mediate interactions with cells and vasculature within the tumor environment. Mutations in the tumor suppressor p53, and the tricarboxylic acid cycle enzymes Isocitrate Dehydrogenase 1 and 2 have been implicated in oncogenic signaling as well as establishing metabolic phenotypes in genetically-defined subsets of malignant glioma. These pathways critically contribute to tumor biology. The aim of this review is two-fold. Firstly, we present the current state of knowledge regarding the metabolic strategies employed by malignant glioma cells, including aerobic glycolysis; the pentose phosphate pathway; one-carbon metabolism; the tricarboxylic acid cycle, which is central to amino acid metabolism; oxidative phosphorylation; and fatty acid metabolism, which significantly contributes to energy production in glioma cells. Secondly, we highlight processes  which are understood to link bio-energetic pathways with oncogenic signals, thereby allowing the glioma cell to achieve a pro-malignant state. Glioma is the most common form of adult-onset primary malignant brain tumor, with 5 cases per 100,000 people diagnosed each year CBTRUS, . In apprGenetically-defined subtypes of GBM have been identified through large-scale analysis of patient tissue samples . There is a great need to develop new therapies, to improve overall survival time and quality of life for these patients. Recent efforts in drug development for slowing glioma progression has focused on the inhibition of growth factor receptor tyrosine kinases, cell-surface receptors, and pro-malignancy kinase signaling pathways. Cancer cell metabolism also provides ample scope for the identification of new therapeutic targets. This review aims to comprehensively summarize the current state of knowledge regarding glioma cell metabolism, as well as the open questions in this fast-moving field.The field of cancer cell metabolism has exploded in the past ten years, but why now? Recently, it has become appreciated that classical model systems for studying malignant glioma may not perfectly reproduce the biochemistry or physiology of human tumors. Two critical factors have greatly impacted the recent boom in this research area: the development of new cell culture techniques (particularly neurospheres and serum-free adherent primary cultures) and new animal models of malignant glioma, including patient-derived xenograft (PDX) models, genetically-engineered mouse (GEM) models, and syngeneic transplant models.Cell culture is a critical tool in our field, but care must be taken in the process of cell isolation and maintenance. Unfortunately, genetic profiling has undermined confidence in the integrity of older cell lines. For example, the sub-clone of U-87 commonly used in many labs today (U-87-MG) is not actually derived from the original tumor cells, although it probably is derived from a human glioblastoma  cycle Figure . This prThe advantage that the Warburg effect conveys to cancer cells is unclear. It has been posited that ATP levels are not a limiting factor for cancer cells  are less glycolytic than differentiated glioma cells, consuming lower levels of glucose and producing lower amounts of lactate while maintaining higher ATP levels compared with their differentiated progeny. The notorious radio-resistance of this cell population is correlated with higher mitochondrial reserve capacity, leading the authors to conclude that GSCs primarily rely upon oxidative metabolic strategies and will not be vanquished by therapies aiming to inhibit glycolysis , despite being detected by contrast MRI  and the structural elements of secondary messengers . To accomplish this metabolic task, glucose-6-phosphate produced during the initial steps of glycolysis is diverted through the pentose phosphate pathway (PPP), where it is converted to ribose-5-phosphate Figure . With thThe PPP appears to be particularly active in dividing cells within glioma. Telomerase reverse transcriptase (TERT), which is necessary for maintenance of dividing cells, is associated with increased expression and phospho-activation of PPP enzymes through the generation of reactive oxygen species (ROS) . Some studies using cultured cell lines have suggested that glioma cells use glutamine as a catabolic substrate, entering the Kreb's Cycle through IDH  and IDH2 (R172K) catalyze the conversion of α-ketoglutarate into the oncometabolite 2-hydroxyglutarate. Interestingly, the evolutionarily-distinct IDH3, which produces NADH not NADPH, does not appear to be mutated at any appreciable rate in glioma cells    can predispose cells to oncogenic transformation and cyst formation leading to renal cancer and renal cysts, respectively  and SDH (Complex II) enzyme expression in more hypoxic areas of the tumor. More recently, one group observed significantly lower Complex II-IV activity in anaplastic astrocytomas and lower Complex I-IV activity in glioblastomas compared with normal brain tissue, using dissociated cells from freshly-frozen human tumors , it is useful to identify whether mitochondrial DNA (mtDNA) itself is intact. Upon disuse in highly glycolytic cells, genetic drift occurs and the mitochondrial genome accumulates mutations, so mtDNA integrity is a good indicator of mitochondrial enzyme function , then imported into the mitochondria for beta-oxidation . Glioma cells contain FASN, and indeed the expression of this enzyme increases with tumor malignancy , are non-specific , sponsored by the Johann Wolfgang Goethe University Hospital in collaboration with TAVARLIN.Pilot Study of a Metabolic Nutritional Therapy for the Management of Primary Brain Tumors (Ketones), sponsored by Michigan State University in collaboration with Sparrow Health System.Ketogenic Diet as Adjunctive Treatment in Refractory/End-stage Glioblastoma Multiforme: a Pilot Study, sponsored by Mid-Atlantic Epilepsy and Sleep Center in collaboration with University of Pittsburgh.Ketogenic Diet With Radiation and Chemotherapy for Newly Diagnosed Glioblastoma, sponsored by St. Joseph's Hospital and Medical Center Phoenix.It is important to note that catabolic pathways do not exist in isolation; they are inextricably linked to the biology of the cancer cell Figure . SeveralBio-energetic pathways in glioma cells do not occur in isolation; importantly, they are connected to each other through cross-signaling. A case in point is the Randle Effect, a prime example of tightly-coordinated cellular energy metabolism which provides a mechanistic link between beta-oxidation and aerobic glycolysis.In the 1960s and 1970s, Randle showed that NADH and acetyl-CoA produced during beta-oxidation both inhibit the activity of pyruvate dehydrogenase (PDH), thereby promoting the conversion of pyruvate to lactate. Well-characterized in diabetes, the work of Randle and his colleagues reveals that non-oxidative glycolysis can occur alongside the oxidation of other substrates, particularly fatty acids ; NADH and ATP produced in the course of beta-oxidation lead to activation of PDK, which in turn phospho-inactivates PDC, leading to lower rates of glucose oxidation and higher rates of lactate release  may seem paradoxical. Yet futile cycles accomplish two important tasks: Firstly, they ensure the continuous availability of raw materials through biosynthesis , while providing a good source of ATP and NADH through the catabolism of any excess material; and secondly, they are thought to play a critical role in regulating metabolic processes, as a futile cycle can induce a bi-stable oscillatory state which is highly sensitive to small changes in enzymatic activity and can be used to communicate changes within the cell  is the central energy sensor within all mammalian cells and consists of three subunits: the catalytic α-subunit and two regulatory β- and γ-subunits  motifs, which have the ability to bind ATP, AMP and ADP in accordance with their cellular ratios  is the major activator of AMPK and constitutively phosphorylates Thr172, however high ATP binding promotes its rapid dephosphorylation , which regulate fatty acid and cholesterol metabolism respectively  can augment mTOR activity, resulting in glioma development  GTPase activity. Upon mitogenic signaling the TSC1/2 complex is stabilized, allowing RHEB to freely activate mTORC1 . Within glioma p53 has a varied role, retaining its wild-type conformation in most primary glioblastomas and acquiring gain-of-function mutations in the pro-neural glioblastoma subtype and during lower-grade glioma progression  under stress in HTB-15 human glioblastoma cells  ratios in response to oxidative stress in glioblastoma cells by increasing GSH pre-cursors  correlates with p53 induction of SCO2, which is necessary for complete respiratory chain function in human glioma   are nuclear receptors; the three subtypes  exert different effects on cellular behavior, although broadly do so by impinging on metabolic gene transcription Figure . PPARs aPPARα expression is lower in high-grade gliomas compared with normal brain tissue; signifying worse prognosis in GBM patients. PPARα overexpression inhibits growth, invasion, and aerobic glycolysis in glioma cells  which hydroxylate Pro402 and Pro564 on HIF-1α. This allows for the recruitment of Von Hippel-Lindau ubiquitin E3 ligase which marks HIF-1α for proteasomal degradation Semenza,  Figure . As PHDsDownstream of its regulation HIF-1α acts in concert with HIF-1β as a transcription factor to coordinate metabolic changes to preserve oxygen and induce angiogenesis to increase oxygen tension. HIF-1 regulates a shift from oxidative metabolism to anaerobic glycolysis when oxygen levels are depleted  through Ras to HIF-1α upregulates mRNA encoding IGFBP2, thus autoregulating HIF by decreasing IGF1 signaling  have different metabolic strategies or preferred metabolic substrates? How do glioma cells balance anabolic and catabolic needs to support growth and invasion? What is the best substrate to use as a radioligand for PET imaging in these tumors? And, of course, can these bio-energetic pathways be targeted pharmacologically to slow growth and invasion of glioma? The stage is set to enter a new era in glioma biology, by augmenting our knowledge of genetics and intracellular signaling with a more comprehensive understanding of cellular bio-energetics.MS and ES discussed the ideas and wrote the paper.ES was supported by Newcastle University's Institute of Neuroscience and MS performed this work as part of her degree in Newcastle University's MRes Programme in Medical and Molecular Biosciences.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.


Objective: To determine the effect of renal denervation (RDN) on the severity of atherosclerosis and aortic aneurysm in hypertensive mice.Methods: Hypertension, atherosclerosis and aortic aneurysm were induced by subcutaneous infusion of angiotensin II (1 μg/kg/min) for 28 days in apolipoprotein E-deficient mice. RDN was conducted using combined surgical and local chemical denervation. The norepinephrine concentration in the kidney was measured by high-performance liquid chromatography. Blood pressure was measured by the tail-cuff method. Atherosclerosis was assessed by Sudan IV staining of the aortic arch. The aortic diameter was measured by the morphometric method. The mRNA expression of genes associated with atherosclerosis and aortic aneurysm were analyzed by quantitative PCR.Results: RDN decreased the median norepinephrine content in the kidney by 93.4%  5 days after the procedure, indicating that the RDN procedure was successful. RDN decreased systolic blood pressure in apolipoprotein E-deficient mice. Mice that had RDN had more severe aortic arch atherosclerosis . The severity of the atherosclerosis was negatively correlated with the renal norepinephrine content . RDN did not affect the size of aortic aneurysms formed or the incidence of aortic rupture in mice receiving angiotensin II. RDN significantly increased the aortic mRNA expression of matrix metalloproteinase-2 (MMP-2).Conclusion: RDN promoted atherosclerosis in apolipoprotein E-deficient mice infused with angiotensin II associated with upregulation of MMP-2. The higher MMP-2 expression could be the results of the greater amount of atheroma in the RDN mice. The findings suggest further research is needed to assess potentially deleterious effects of RDN in patients. Renal denervation (RDN) is used in clinical practice to lower blood pressure in treatment-resistant hypertension  mice fed a high-fat diet  were purchased from the Animal Resources Centre, Perth, Australia. All experiments were conducted in a temperature-controlled animal house (21 ± 1°C) under a 12:12-h light-dark cycle and mice were given standard chow and water ad libitum. All animal protocols conformed to the Guide for the Care and use of Laboratory Animals by the United States National Institutes of Health and the Australian Code of Practice for the Care and Use of Animals for Scientific Purpose . Institutional ethics approval was obtained from both James Cook University and Federation University Australia.Male ApoE−/− mice underwent sham surgery and seven mice underwent bilateral RDN. Five days later, the mice were euthanized and the right kidney was collected and norepinephrine content determined.A preliminary study was carried out to confirm the success of the RDN procedure. Five ApoE−/− mice underwent bilateral RDN and 20 ApoE−/− mice underwent sham surgery 1 day after the baseline blood pressure was measured . This method had a good reproducibility with an inter-assay coefficient of variation of 4.7% (n = 10).The right kidney was homogenized in 1 mM ethylenediaminetetraacetic acid (EDTA) and 4 mM sodium metabisulfite. The norepinephrine content in the homogenate was measured by phase isocratic high-performance liquid chromatography (HPLC)   in Eppendorf tubes according to the manufacturer's guidelines. The RNA yield of one sample from the sham surgery group was too low and not sufficient for the experiment, and thus this sample was not used for the subsequent cDNA synthesis and quantitative PCR. RNA was reverse transcribed to cDNA using the High Capacity Reverse Transcription Kit (Life Technologies). Gene expression was assessed by quantitative PCR using SYBR reagents. Primer sets are outlined in Table Blood was collected by cardiac puncture at the time of mice sacrifice. The concentration of total cholesterol, low-density lipoprotein/very low-density lipoprotein (LDL/VLDL) cholesterol and high-density lipoprotein (HDL) cholesterol in the plasma were quantified using a commercial available kit   model using S Plus (software version 8.2). The difference in survival between the two groups was analyzed using log rank test. The correlation between atherosclerosis severity and renal norepinephrine content was assessed using the correlation analysis function of the GraphPad Prism 5 software . Differences were considered to be statistically significant at P < 0.05.Continuous numbers were presented as a median and interquartile range (IQR). The difference between two groups was analyzed using Mann-Whitney P > 0.05, Table The baseline body weight, systolic, diastolic and mean blood pressure, and the heart rate were similar in the sham surgery and the RDN groups  was lower in the RDN group compared with sham surgery group , inducible nitric oxide synthase (iNOS), monocyte chemoattractant protein-1 (MCP-1), nuclear factor-kappa B (NF-κB), tumor necrosis factor-alpha (TNF-α) and matrix metalloproteinase-9 (MMP-9) within the thoracic aorta were similar in mice receiving RDN and sham surgery Table . Mice remRNA expression of angiotensin receptors (both type 1 and 2) and α2 adrenoceptors in the thoracic aorta were similar in mice that received RDN and sham surgery Table .P > 0.05, Figure RDN did not affect plasma levels of LDL/VLDL cholesterol, HDL cholesterol, or total cholesterol (This study focussed on assessing the effect of RDN on atherosclerosis severity in a mouse model. RDN was successful performed as evidenced by substantially lower norepinephrine content of the kidney 5 and 33 days after the procedure and lower systolic blood pressure in mice having RDN compared to controls. The main finding of the study was that the severity of aortic arch atherosclerosis, as assessed by Sudan IV staining area, was greater in mice that received RDN than controls. The severity of atherosclerosis was correlated with the extent of the RDN, as assessed by renal norepinephrine levels. Our results suggest that RDN did not affect aortic aneurysm severity or rupture.A number of previous experimental studies have reported that sympathetic denervation promoted atherosclerosis at sites remote to the denervation procedure (Murphy et al., The mechanisms underlying the atherosclerosis-promoting effect of sympathetic denervation are not well understood. It has been reported that this effect of sympathetic denervation may be due to stimulating migration of adventitial fibroblasts to the media and the associated loss and dedifferentiation of smooth muscle cells (Kacem and Sercombe, −/− mice fed a high-fat diet for 10 weeks (Wang et al., Our results are in contrast to the results of a recent report which used another atherosclerotic model, i.e., ApoE−/− mice fed a high-fat diet (Johnson et al., MMP-2 expression within the thoracic aorta was greater in mice receiving RDN than controls. MMP-2 plays an important role in degrading extracellular matrix and has been implicated in the initiation, development and eventual rupture of atherosclerotic plaques (Li et al., Other possible explanation for the greater atherosclerosis in mice receiving RDN include increased arterial pressure variability or functional changes within the media. These have not been investigated in the current study.RDN has been reported to promote renal artery stenosis in humans. The Symplicity HTN studies (Krum et al., Renal denervation has been shown to decrease blood pressure in a large number of clinical (Krum et al., This study has several limitations. First, the sample size of the study was small; second, this study employed only one animal model; third, blood pressure was measured by the tail-cuff method rather than the gold standard telemetry method; and fourth, the RDN procedure in mice is different from the clinical practice in which catheter-based methods are used. Finally the plasma concentrations of lipids varied substantially in different mice and the reasons for this are not clear.Based on our results and other experimental evidence (Murphy et al., All authors listed, have made substantial, direct and intellectual contribution to the work, and approved it for publication.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.