Selected response items and constructed response (CR) items are often found in the same test. Conventional psychometric models for these two types of items typically focus on using the scores for correctness of the responses. Recent research suggests, however, that more information may be available from the CR items than just scores for correctness. In this study, we describe an approach in which a statistical topic model along with a diagnostic classification model (DCM) was applied to a mixed item format formative test of English and Language Arts. The DCM was used to estimate students’ mastery status of reading skills. These mastery statuses were then included in a topic model as covariates to predict students’ use of each of the latent topics in their written answers to a CR item. This approach enabled investigation of the effects of mastery status of reading skills on writing patterns. Results indicated that one of the skills, Integration of Knowledge and Ideas, helped detect and explain students’ writing patterns with respect to students’ use of individual topics. Selected response  items and constructed response  items are often found in the same test. An important benefit of SR items is their efficiency in being scored quickly with minimal potential for raters’ bias. CR items, on the other hand, have been shown to be appropriate for assessing certain types of higher order knowledge, as this type of item can be used to require students to construct their answers and frequently show their reasoning in their answers .While SR and CR items are used together, existing psychometric approaches do not benefit from both data sources efficiently. Most psychometric models, including item response theory models and diagnostic classification models (DCMs), have been developed for focusing on item scores, i.e., correctness of the responses. This is true for CR items as well. The partial credit model  and the Statistical topic models , on the In this study, we present an approach in which results from a DCM were used in a topic model as covariates to understand the relationship between students’ mastery status of reading skills and the latent thematic structure in students’ writing to answer to a CR item. Specifically, a log-linear cognitive diagnostic model LCDM;  was usedDiagnostic classification models  are probj) parameters and the mastery status of the individual (i) given the Q-matrix as follows  is a vector of linear combinations of the αi and qj, which specifies an effect structure of the model. h can include main effect of each attribute, two-way interactions, three-way interactions, etc., depending on how many attributes there exist in the test. For instance, if the effect structure includes only main effects and two-way interactions, the model can be represented aswhere λjs represents the main effects of attribute s on item j and λjsu represents the two-way interaction effects between the combination of attributes s and u on item j. As indicated earlier, this can be extended to three-way or more interaction terms, if needed. Due to the flexibility of this effect structure, the LCDM provides a general framework for DCMs. Further, one can investigate whether the relationship among attributes is compensatory or non-compensatory. For example, using a significance test for λjsu without predetermining the magnitude of the relationship of the two attributes s and u on item j, the relationship between attributes s and u on item j can be tested.where λTopic models are statistical models designed to extract the latent topic structure in a collection of documents . Latent –d:For each document, оd)∼LogisticNormalDraw the topic proportions for the document ]For each word in the document, [оzd,n)∼Multinomial (θd)Draw word’s topic assignment ∼Multinomial Conditioning on the topic chosen, draw an observed word from that topic  follow normal distributions . The inclusion of one or more covariates allows the model to borrow strength from documents with similar covariate values for estimating the document proportion  test. The test was designed to provide formative information on how well students understood concepts and could demonstrate their knowledge in reading and writing.The test consisted of five items: three multiple choice items, one short answer (SA) item, and one extended response (ER) item to measure reading and writing ability. Two scores were assigned for the ER item. A confirmatory factor analysis supported this two-factor model: the multiple choice and SA items formed one factor, reading ability, and the two scores for the ER item measured the other factor, writing ability. A non-linear internal consistency estimate  for thisIdea), identifying the structure of a text (Structure), and integrating knowledge of ideas (Integration). These three skills were used to create the entries in the Q-matrix shown in Mplus version 7.4 , but, or, etc. Stemming consists of converting words to their root form. For instance, all verbs were converted to the present tense, plural forms were converted to singular form, words that have similar morphology  were converted to a root form such as do, and typographical errors were corrected.The STM topic model was used to identify latent topics in students’ written responses to the ER item and investigate the relationship between reading and writing ability. The first step in applying any topic model is to preprocess the text. This is done to help the estimation process and improve interpretability of the resulting model . PreprocAfter stemming words and removing stopwords, words with a frequency of less than 10 and documents with less than 15 words were excluded. In addition, documents with a score of 0 were excluded as this indicated the responses were not scorable. As shown in stm , but the intercepts for items 2 and 4 were not. Idea and Structure was 0.86, the correlation between Idea and Integration was 0.67, and the correlation between Structure and Integration was 0.57. These indicated substantial relationships between skills. Eight different mastery profiles are possible for the three skills in the Q-matrix. Results in Integration , students who had mastered Idea and Integration , and students who had mastered all three skills . Students’ mastery statuses for each attribute obtained by this analysis were included in the STM as covariates to predict the use of topics.For item 4, as no significant interaction effect for attributes 1 and 3, the interaction term was dropped from the effect structure in the final LCDM model. To detect the number of topics, STM models with from 2 to 20 topics were fit to the data as an exploratory analysis. As described in Methods section, semantic coherence, exclusivity, and cosine similarity were used to determine the number of topics. Thebold and underlined words are the highest frequency words forthe given topics.One way to help interpret and characterize each topic in the model is to examine (1) written responses of students who were the highest probability users of each topic and (2) the highest probability words for each topic. The 15 highest probability words in each topic for the four-topic STM are listed in pollution, paper, mill, industry, coastal, and water . These words come from the prompt . Students had been instructed to use information from the passages to support their arguments. This topic was labeled Integrative Borrowing as it reflected this use of the terms in the prompt. The following is the answer of the student who was the most frequent user of words from this topic.In the first topic, the highest frequency words were Paper mills are having a negative effect. Passage A says “Paper mills are the third largest polluters in the United States., releasing pollutants into the air, water, and soil.” Passage A also say that “many paper mills are working to reduce the amount of pollutants they produce today.” But they are letting it out, and it also effecting in passage B it says that “the fishing industry decreases due to pollution caused by paper mills.” That why I think paper mills are having a negative effect.(Integrative Borrowing) tree, animal, cut, and people. These words reflect use of everyday language but not directly related to the question. This topic was labeled as Everyday Language. The following is the answer of a student who was a high frequency user of words from topic 2.The highest frequency words in the second topic were animals homes away so how would you feel if someone just took your house away and built something else and just put your family out on the street with nowhere to go, that’s how the animals feel. Your destroying our plants life to that we need the plants and animals were there first and they really don’t have any other home to go to besides a zoo why do that when they can just be free without the people harming them.(Everyday Language) No because they are killing all the plants and taking the Georgia, timber, acre, coastal, plain, and pine). . The words in the fourth topic were copied from the stem . The followings are answers of students who were the highest frequency users of words from the third and the fourth topics, respectively. Characteristic of users of topics 3 and 4 is that these words were simply copied from the passage or stem without any clear effort to integrate the words into the argument.The highest frequency words in the third and fourth topics were both borrowed directly from either the stem or the passages. The words in the third topic were copied from the passages  I think that the small protected forest should not be developed into commercial representative should not allow the protected forest to be developed into the commercial timberland. They shouldn’t because, in passage B it states that the soil isn’t suitable for any kind of forest. The timberland is worth an average of $97 a year because the land isn’t suitable for the tree’s and soil. That is why you shouldn’t allow them to put the protected forest in the timberland.(Copied from Stem) The Integrative Borrowing in their answers tended to have higher scores and (2) students who used more Everyday Language in their answers tended to have lower scores.Key Ideas or Craft and Structure did not have a significant impact on students’ use of the topic. Integration of Knowledge and Ideas (Integration) was the only skill that had a significant effect on the use of each topic at p < 0.05. As seen in Integrative Borrowing and Everyday Language tended to be related to the rubric based score. The results in Integration, their probability of using the integrative borrowing topic increases by 0.07, their probability of using the copying from passage topic increases by 0.05, their probability of using the copying from stem topic increases by 0.05, but their probability of using the everyday language topic decreases by 0.15.The results indicate mastery status of either Integration of Knowledge and Ideas was the pivotal skill for the use of each of the four topics. That is, as students mastered Integration of Knowledge and Ideas, they tended to use more of the Integrative Borrowing topic in their writing and less of the Everyday Language topic. CR or ER items are often used to assess higher-order thinking skills. Rubric-based scores provide useful information regarding students’ knowledge status with respect to the objectives being measured on the test. There is also information about students’ thinking and reasoning as reflected in their answers, however, that can be missed by the rubric-based scores alone  were included as covariates to predict students’ use of the topics. Although three skills were identified in the Q-matrix, only four of the eight possible mastery profiles were present in the data. The four-topic STM was found to be the best fit to the textual data from the students’ answers to the test questions along with students’ reading skills as covariates. The results showed that mastery status of es alone . For exaes alone  or writiThe assessment used in this study was a formative assessment and was not specifically designed to fit a DCM model. Due to the small number of items in the assessment, the skill reliabilities were relatively low, which is a possible limitation of this study. Even with this limitation, however, results demonstrate that combining results from a DCM with a topic model enables the possibility of investigating the relationship between the knowledge as measured by the multiple choice items and cognitive skills used in answering to the CR items. Topic modeling is relatively new in educational research, but it has been found to provide a useful set of methodological tools for extracting this added information in the text of answers to CR items.Some of current techniques developed in natural language processing or machine learning may not be applicable for the text in education as the text in education may have different characteristics from the text in social networks or publications. Further studies would be helpful to address important issues in this area, such as what could be the effects of stemming methods on latent topic structure or what methods could be used for selecting the best fitting topic model.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Human Subjects Office, University of Georgia. Written informed consent from the participants’ legal guardian/next of kin was not required to participate in this study in accordance with the national legislation and the institutional requirements.All authors listed have made a 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.


Dcc haploinsufficiency within dopamine neurons in adolescence. Dopamine targeting in adolescence requires the complementary interaction between DCC receptors in mesolimbic dopamine axons and Netrin-1 in the NAcc. Factors regulating either DCC or Netrin-1 in adolescence can disrupt mesocorticolimbic dopamine development, rendering vulnerability or protection to phenotypes associated with psychiatric disorders.The fine arrangement of neuronal connectivity during development involves the coordinated action of guidance cues and their receptors. In adolescence, the dopamine circuitry is still developing, with mesolimbic dopamine axons undergoing target-recognition events in the nucleus accumbens (NAcc), while mesocortical projections continue to grow toward the prefrontal cortex (PFC) until adulthood. This segregation of mesolimbic versus mesocortical dopamine pathways is mediated by the guidance cue receptor DCC, which signals dopamine axons intended to innervate the NAcc to recognize this region as their final target. Whether DCC-dependent mesolimbic dopamine axon targeting in adolescence requires the action of its ligand, Netrin-1, is unknown. Here we combined shRNA strategies, quantitative analysis of pre- and post-synaptic markers of neuronal connectivity, and pharmacological manipulations to address this question. Similar to DCC levels in the ventral tegmental area, Netrin-1 expression in the NAcc is dynamic across postnatal life, transitioning from high to low expression across adolescence. Silencing Netrin-1 in the NAcc in adolescence results in an increase in the expanse of the dopamine input to the PFC in adulthood, with a corresponding increase in the number of presynaptic dopamine sites. This manipulation also results in altered dendritic spine density and morphology of medium spiny neurons in the NAcc in adulthood and in reduced sensitivity to the behavioral activating effects of the stimulant drug of abuse, amphetamine. These cellular and behavioral effects mirror those induced by  Adolescence is a critical developmental period characterized by dramatic neurological and behavioral changes, with neurocircuitry in the prefrontal cortex (PFC), a brain region essential for cognitive and reward functions, being established during this time . The devNetrin-1 haploinsufficiency mice. Adult mice with Netrin-1 haploinsufficiency show increased medial PFC dopamine concentrations in comparison to wild-type mice and are protected against amphetamine-induced increase in locomotor activity similarly to adult mice with Dcc haploinsufficiency  with food and water available ad libitum. All the experiments were conducted during the light part of the cycle.d-Amphetamine sulfate  was dissolved in 0.9% saline and administered i.p. at a volume of 0.1 ml/10 g and at a dose of 2.5 mg/kg of amphetamine.Mice were rapidly decapitated, and their brains were flash-frozen in 2-methylbutane  chilled with dry ice. Bilateral punches of the NAcc were excised from 1-mm-thick coronal slices starting from sections corresponding to Plate 14 of the mouse brain atlas  and procNetrin-1 were used . Three scrambled controls were created by rearranging the sequence order so that there was less than a 64% interaction rate. Active or control shRNA sequences were cloned into a pLentiLox 3.7 vector . Importantly, the constructs express GFP under the CMV promoter and the shRNA sequence under the U6 promoter, which allows verification of infection and visualization of the injection site for the in vivo experiments. The efficiency of knockdown for shRNA constructs was tested in cultured HEK cells by co-expressing a pBKNetrin-1-Flag plasmid containing the mouse Netrin-1 cDNA cloned into a pBK-CMV vector (Stratagene) with a C-terminal Flag tag (gift of Dr. Andreas Püschel to HM Cooper), Flag expression was then evaluated by western blot using an anti-Flag antibody . Lentiviruses expressing shRNAs and scrambled controls were prepared by the SPARC Biocentre lentiviral core facility .Pre-designed and validated siRNA sequences (Ambion) were used to create shRNA by the addition of a standard hairpin loop (TTCAAGAGA) between the sense and antisense sequences. Three independent pre-designed and validated siRNA sequences against mouse NAcc coordinates: +2.6 mm (anterior/posterior), +1.5 mm , and −3.75 mm  relative to Bregma, at a 30°. A total of 0.5 μl of purified virus was delivered on each side over an 8-min period followed by a pause of 6 min as previously  21±1 mice were anesthetized with isoflurane (5% for induction and 2% for maintenance) and placed in a stereotaxic apparatus. Simultaneous bilateral microinfusions of the lentivirus expressing an shRNA against Netrin-1 or a scrambled sequence, each with GFP, into the NAcc were performed stereotaxically using Hamilton syringes. eviously . At the Adult mice received an intraperitoneal overdose of ketamine 50 mg/kg, xylazine 5 mg/kg and acepromazine 1 mg/kg and were perfused intracardially with 50 ml of 0.9% saline followed by 75 ml of chilled fixative solution . Brains were dissected and placed in the fixative solution overnight at 4°C and were then transferred to phosphate-buffered saline and stored for a maximum of 2 days. Brains were sectioned using a vibratome .Every second coronal section was processed (1:2 series) as previously reported , 2013. A® (MicroBrightField) (® (MicroBrightField)  , 2013. ThtField) , 2013. Tx = 175 μm, y = 175 μm) from a random start point. Counting of varicosities was performed at ×100 magnification on 5 of the 12 sections contained within the rostrocaudal borders of our region of interest . A guard zone of 5 μm was used and the optical dissector height was set to 10 μm.The medial PFC subregions were delineated according to plates spanning 14–18 of the mouse brain atlas . A 5× ma1. Only dendritic segments from both hemispheres with at least 10 μm of length and that were clearly distinguishable from other segments were included in the analysis. NeuronStudio determines dendrite length semi-automatically and classifies individual spines into thin, mushroom, or stubby according to (i) spine aspect ratios, (ii) head-to-neck diameter ratios, and (iii) head diameters. Thin spines have a neck ratio value (head to neck diameter ratio) less than 1.1 and a length to spine head diameter greater than 2.0. Mushroom spines have a neck ratio value above 1.1 and a spine head diameter equal or greater than 0.3 μm. Stubby spines were discernable by the lack of neck. Each dendritic segment was analyzed separately according to the number of spines and the diameter of the head spines. The density and head diameter were calculated per 10 μm of dendritic length. The analysis was performed by an experimenter fully blinded across groups.Dendrites from GFP-positive neurons contained in the NAcc were systematically selected for imaging fluorescence, using a confocal microscope (Olympus FV 1200) at 60× immersion objective at 4× zoom and 1024 × 1024 pixels resolution. The Z stacks acquisition were performed at 0.3 μm increments. For each mouse, approximately 5–8 dendritic segments were quantified using NeuronStudio softwareLocomotor activity was quantified as before  using ant-tests. All data are normally distributed, and the variance is similar between groups. Statistical differences between more than two groups were analyzed with one-way or two-way ANOVAs, followed by Bonferroni multiple comparison post hoc tests. The sample size in all the experiments varied from 4 to 8 animals per group.All statistical analyses were performed using Prism 6 for Windows . All values are represented as means ± S.E.M. A significance threshold of α = 0.05 was used in all the experiments. Statistical differences between two groups were analyzed with Student’s Dcc mRNA and protein expression in the VTA decreases from adolescence to adulthood  = 5.20, p = 0.014; the expression at PND21 is significantly higher than PND75; post hoc Bonferroni, p < 0.05). The similarity between Netrin-1  = 3.50, p = 0.06, adapted from We have shown previously that dulthood . If Netrdulthood  and founNetrin-1  and DCC Netrin-1 (Netrin-1 shRNA 1–3) and their corresponding scrambled sequences (Scrambled 1–3) using cultured HEK cells that co-expressed a mouse Netrin-1 protein with a C-terminal Flag tag .We tested the efficiency of three different viral constructs expressing shRNA sequences against Flag tag . InfectiNetrin-1 shRNA”) into a lentivirus and injected it bilaterally into the NAcc of P21 mice. Control mice received bilateral microinfusions of the corresponding scrambled sequence  = 3.76, p = 0.007). This effect is transient because Netrin-1 protein expression in the NAcc of adult mice that received Netrin-1 shRNA or scrambled construct microinfusions in early adolescence do not differ (t(8) = 1.25, p = 0.25).We packaged the shRNA construct 2  = 9.09, p = 0.0195; no significant virus microinfusion × medial PFC region interaction, F = 0.117, p = 0.89; significant main effect of medial PFC region, F = 139.1, p < 0.0001). This effect is accompanied by a significant increase in the total number of dopamine varicosities in Netrin-1 shRNA mice versus controls  = 14.23, p = 0.007; no significant virus microinfusion × medial PFC region interaction, F = 0.173, p = 0.21; significant main effect of medial PFC region, F = 118.5, p < 0.0001, post hoc Bonferroni, significant difference in the prelimbic subregion between Netrin-1 shRNA- and Scrambled-infused mice, p = 0.002). There are no differences between Netrin-1 shRNA and scrambled groups in the density of PFC TH-positive varicosities  = 0.63, p = 0.45; no significant virus microinfusion × medial PFC region interaction, F = 0.58, p = 0.57; no significant main effect of medial PFC region, F = 1.10, p = 0.36). These anatomical changes are similar to those observed in adult mice with downregulation of DCC receptors in dopamine axons in adolescence, suggesting that Netrin-1 signaling through DCC receptors is required for mesolimbic dopamine axon targeting in the NAcc  = 2.04, p = 0.048) and there is a significant reduction in the density of mushroom spines in the Netrin-1 shRNA group compared to Scrambled-infused mice  = 4.26, p = 0.045; significant virus microinfusion × spine type interaction, F = 4.38, p = 0.016; significant main effect of spine type, F = 29.86, p < 0.0001, post hoc Bonferroni, significant difference in mushroom spines between Netrin-1 shRNA- and Scrambled-infused mice, p = 0.0038). While not significant, we also observed a trend toward a reduction in the total head diameter (t(41) = 1.82, p = 0.076).Netrin-1 promotes synapse formation in the PFC  and potec at P21 . Total dDcc expression in dopamine neurons in adolescence leads to blunted locomotor response to amphetamine in adulthood  = 0.80, p = 0.38, no significant virus microinfusion × time interaction: F = 0.79, p = 0.70; significant main effect of time: F = 13.8, p < 0.0001; stereotype counts (inset): no significant main effect of virus microinfusion: F = 3.78, p = 0.066, significant virus microinfusion × time interaction: F = 2.02, p = 0.010, significant main effect of time: F = 5.09, p < 0.0001). However, mice that had received Netrin-1 shRNA infusions in adolescence show reduced amphetamine-induced locomotion and stereotypy counts in adulthood, when compared to Scrambled controls  = 4.92, p = 0.038, significant virus microinfusion × time interaction: F = 2.13, p = 0.006; significant main effect of time: F = 8.55, p < 0.0001; stereotype counts (inset): no significant main effect of virus microinfusion: F = 3.78, p = 0.066, significant virus microinfusion × time interaction: F = 2.02, p = 0.010, significant main effect of time: F = 5.09, p < 0.0001).We have shown that reduced dulthood , and thadulthood . Here weDcc haploinsufficiency, strongly suggesting that the interaction between DCC receptors in dopamine axons and Netrin-1 in the NAcc is required for the appropriate establishment of mesocorticolimbic dopamine circuitry in adolescence.The fine arrangement of neuronal connectivity during development involves the coordinated action of guidance cues and their receptors. DCC receptors within VTA dopamine neurons are crucial for the targeting of their mesolimbic projections to the NAcc in adolescence and for delimiting the extent of their input to the PFC . Here weWe have previously observed a high-to-low complementary expression of Netrin-1 in the NAcc and of DCC receptors in mesolimbic dopamine axons during adolescence and adulthood that suggested a direct interaction of DCC and Netrin-1 signaling . The simSpines of MSN in the NAcc emerge after the second postnatal week, with peak spine density occurring around the third postnatal week in rodents . Netrin-The overall reduction in spine density after Netrin-1 downregulation appears to be driven by a reduction in the density of mushroom spines, which are required for synapse maturation and stabilization . MushrooNetrin-1 shRNA in adolescence show diminished locomotor response to a challenge injection of the stimulant drug amphetamine. The increase in PFC dopamine connectivity is likely underlying these behavioral effects, considering that mesocortical dopamine neurotransmission exerts inhibitory control over mesolimbic dopamine activity (Netrin-1 shRNA likely prevents amphetamine-induced dopamine release into the NAcc, resulting in the observed blunted drug locomotor response. This idea is consistent with the behavioral phenotype displayed by adult mice with Netrin-1 or with Dcc haploinsufficiency (Netrin-1 shRNA infusions in adolescence. MSN receive multiple excitatory synaptic inputs via their numerous spines. A reduction in spine density and altered shape may render MSNs less excitable, affecting NAcc-mediated behaviors, including drug-induced locomotor activity. Indeed, dopamine and excitatory inputs converge onto MSN and mediate behavioral and incentive motivational effects of amphetamine and other psychostimulants (Adult mice microinfused with the activity . The incficiency . These mficiency . We cannimulants .In summary, our findings indicate that the complementary interaction between DCC receptors expressed by dopamine axons and Netrin-1 expressed by NAcc neurons and located in the surrounding extracellular matrix is required for the developmental organization of the mesocorticolimbic dopamine circuitry. Environmental risk or protective factors for psychopathology may impact the development of this system by regulating DCC and/or Netrin-1 expression in adolescence. Adolescent exposure to recreational-like doses of amphetamine  downregThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation, to any qualified researcher.All the animals experiments and procedures were performed according to the guidelines of the Canadian Council of Animal Care and the McGill University/Douglas Mental Health University Institute Animal Care Committee.SC and CF conceived and designed the experiments, analyzed the results, and wrote the manuscript. SC performed all the molecular evaluations, the developmental characterization, drug experiments, and performed stereotaxic surgeries. DN performed all the neuroanatomical analysis. LR and AW developed the different plasmid constructs. LR provided technical training for anatomical analysis and contributed to the editing of the manuscript. AM performed stereotaxic surgeries. AT-B contributed in tissue collection. GH contributed to the writing and editing of the manuscript. HC generated the lentiviral constructs and provided technical training necessary to perform the research. All authors contributed to the article and approved the submitted version.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.


We find that synchronous dopamine secretion is abolished in acute brain slices of conditional knockout mice in which Synaptotagmin-1 is removed from dopamine neurons. This indicates that Synaptotagmin-1 is the Ca2+ sensor for fast dopamine release. Remarkably, dopamine release induced by strong depolarization and asynchronous release during stimulus trains are unaffected by Synaptotagmin-1 knockout. Microdialysis further reveals that these modes and action potential-independent release provide significant amounts of extracellular dopamine in vivo. We propose that the molecular machinery for dopamine secretion has evolved to support fast and slow signaling modes, with fast release requiring the Ca2+ sensor Synaptotagmin-1.Dopamine powerfully controls neural circuits through neuromodulation. In the vertebrate striatum, dopamine adjusts cellular functions to regulate behaviors across broad time scales, but how the dopamine secretory system is built to support fast and slow neuromodulation is not known. Here, we set out to identify Ca Dopamine is an important neuromodulator in the vertebrate brain, but the secretory biology of dopamine is not well understood. A key dopamine pathway arises from midbrain dopamine neurons located in the substantia nigra pars compacta. Their axons send projections to the dorsal striatum, where dopamine neuromodulation controls initiation and execution of movement. A prominent model is that dopamine operates slowly and on distant receptors through volume transmission . Recent 2+-triggering of secretion is the presence of Ca2+ sensors. Various Ca2+ binding proteins are used as Ca2+ sensors for vesicular exocytosis  in acute brain slices. We generated mice that express channelrhodopsin-2 (ChR2) selectively in dopamine neurons using mouse genetics  and the active zone protein Bassoon  . This meDA mice , indicatDA mice , which inhibits action potential firing . Remarka release . Hence, 2+ sensor Synaptotagmin-1. Given the prevailing model of volume transmission, which postulates that dopamine neurotransmission is slow and imprecise, it is surprising that dopamine neurons have evolved to employ a fast Ca2+ sensor. This finding, however, is in line with a recent description of sparse secretory hotspots in striatal dopamine axons Bkmm/J). Mice for the Cre-dependent expression of ChR2 26Sortm32(CAG-COP4*H134R/EYFP)Hze), obtained from the Jackson Laboratories, were crossed to DATIRES-Cre mice, and mice used for experiments were heterozygote for both DATIRES-Cre and ChR2. The conditional Synaptotagmin-1 knockout mice were generated by EUCOMM Wtsi/WtsiCnrm)  , describDA mice striatal dopamine fibers were activated after transduction of dopamine neurons with AAVs for Cre-dependent expression of oChIEF-citrine (Addgene_50973). AAVs (serotype AAV2/5) were generated in HEK293T cells  using calcium phosphate transfection. 72 hr after transfection, cells were collected, lysed, and viral particles were extracted and purified from the 40% layer after iodixanol gradient ultracentrifugation. Quantitative rtPCR was used to measure the genomic titer (2.31–2.75 × 1012 genome copies/ml). For stereotaxic surgeries, mice were anesthetized using 5% isoflurane and mounted on a stereotaxic frame. 1.5–2% isoflurane was used to maintain a stable anesthesia during the surgery. 1 μl of viral solution was injected unilaterally into the right substantia nigra pars compacta  of Syt-1 control and Syt-1 cKODA mice at P25-29 using a microinjector  at the rate of 100 nl/min. After surgery, the mice obtained analgesia and were allowed to recover for at least 21 d prior to recording. Stereotaxic surgeries were performed according to protocols approved by the Harvard University Animal Care and Use Committee.In Syt-1 control and Syt-1 cKO-citrine , a fast 4, 26.2 NaHCO3, 1 NaH2PO4, 12 glucose, 1 sodium ascorbate, 1 myo-inositol, 3 sodium pyruvate, . Slices were incubated at room temperature for 1 hr in incubation solution bubbled with 95% O2 and 5% CO2 containing (in mM): 126 NaCl, 2.5 KCl, 1.3 MgSO4, 2 CaCl2, 26.2 NaHCO3, 1 NaH2PO4, 12 glucose, 1 sodium ascorbate, 1 myo-inositol, 3 sodium pyruvate . Recording was done at 34–36°C, and slices were continuously perfused with artificial cerebrospinal fluid (ACSF) at 3–4 ml/min bubbled with 95% O2 and 5% CO2. ACSF contained (in mM): 126 NaCl, 2.5 KCl, 2 CaCl2 (unless noted otherwise), 1.3 MgSO4, 1 NaH2PO4, 12 glucose, 26.2 NaHCO3, pH 7.4, 300–310 mOsm. Recordings were completed within 5 hr of slicing. In Male and female mice (42–113 days old) were deeply anesthetized with isoflurane and decapitated. 250 µm thick sagittal brain sections containing the striatum were cut using a vibratome  in ice-cold cutting solution with (in mM): 75 NaCl, 75 sucrose, 2.5 KCl, 7.5 MgSODA littermate pair. CFEs were held at 600 mV and placed 20–60 µm below the slice surface in the dorsolateral striatum. Signals were sampled at 10 kHz and low-pass filtered at 400 Hz. Dopamine release was evoked by electrical or optogenetic stimulation every 2 min.For all carbon fiber amperometry, carbon fiber microelectrodes  were made from carbon fiber filaments (Goodfellow). Each CFE was calibrated by puffing freshly made dopamine solutions of increasing concentrations  in ACSF for 10 s . The curst stimulus for 2.935 s and expressed as μM x s after removal of the stimulus artefacts. The total increase (nomifensine – areabaseline), and the fold increase (nomifensine/areabaseline).Optogenetic stimulation was performed with channelrhodopsin expression limited to dopamine neurons by AAV-mediated expression of Cre-dependent oChIEF-citrine  or transincrease  was calcincrease  was calcElectrical stimulation was performed with an ACSF filled glass pipette (tip diameter 3–5 µm) connected to a linear stimulus isolator  to deliver monopolar electrical stimulation (10–90 µA). The stimulation pipette was placed 20–30 µm below the slice surface in the dorsolateral striatum and 100–120 µm away from the tip of the CFE. A biphasic wave (0.25 µs in each phase) was applied to evoke dopamine release. Electrical stimulation was delivered either as a single stimulus or a 10-stimulus 10 Hz train .4, 2 CaCl2, 12 glucose, 10 HEPES, pH 7.3, 300–310 mOsm) was puffed onto the recording site in the dorsolateral striatum for 10 s at 9 µl/s using a syringe pump (World Precision Instruments). Only one KCl puff was applied per slice. The peak amplitude of the dopamine response and the area under the curve were quantified from start of application of KCl to 200 s after the puff, at which time the levels returned to baseline.For KCl stimulation in 2+]ex, CFEs were first tested with a 20 µM dopamine puff at different [Ca2+]ex  to ensure that the CFE correctly reports [dopamine] across variable amounts of [Ca2+]ex .For experiments in variable [Ca[Ca2+]ex . Slices 2, 4 mM,  in separst stimulus in the 10 Hz train before and after TTX was analyzed in Syt-1 control and Syt-1 cKODA mice (Extracellular recordings in ODA mice .DA littermate mice (99–111 days old) were deeply anesthetized with 5% isoflurane and perfused transcardially with ice-cold 30–50 ml phosphate buffer saline (PBS), followed by 50 ml of 4% paraformaldehyde (PFA) in PBS at 4°C. Brains were then left in 4% PFA for 12–16 hr followed by incubation in 30% sucrose + 0.1% sodium azide in PBS overnight or until they sank to the bottom of the tube. Coronal striatal sections (20 µm thick) were cut using a vibratome  in ice-cold PBS. Antigen retrieval was performed on slices overnight at 60⁰C in 150 mM NaCl, 1 mM EDTA, 0.05% Tween 20, 10 mM Tris Base, pH 9.0. After antigen retrieval, slices were washed in PBS, and incubated in Image-iT FX signal enhancer  for 30 min at room temperature. Slices were washed in PBS for 10 min and non-specific binding was blocked in 10% goat serum in 0.25% Triton X-100 in PBS (PBST) for 1 hr at room temperature. Slices were stained with primary antibodies for 12 hr at 4°C, and the following primary antibodies were used: mouse monoclonal IgG2a anti-Bassoon  and guinea pig polyclonal anti-TH . Next, slices were washed three times in PBST for 10 min and incubated in secondary antibodies  for 2 hr at room temperature in PBST. Sections were washed three times in PBST for 10 min and then mounted on Poly-D-lysine coated #1.5 cover glasses  with H-1000 mounting medium (Vectashield). At all times, the experimenter was blind to the genotype of the mice.Male and female Syt-1 control and cKO3) were acquired from 7 to 8 regions within the dorsolateral striatum in each section. For image analysis, regions of interest (ROIs) ranging from 20 × 20 × 2.5 µm3 to 25 × 25 × 2.5 µm3 were selected manually in each image stack. ROIs were analyzed to characterize TH and Bassoon signals and to determine their overlap using a custom written MATLAB code  were applied to each ROI. The volume occupied by TH was quantified and was divided by the total image volume , guinea pig polyclonal anti-TH , and rabbit polyclonal anti-Synaptotagmin-1 antiserum . The secondary antibodies were: goat anti-mouse IgG2a Alexa 488 , goat anti-rabbit Alexa 555 , and goat anti-guinea pig Alexa 633 .Striatal synaptosome preparations were performed as previously described . Wild ty2) stained for Bassoon (detected via Alexa 488), Synaptotagmin-1 (detected via Alexa-555) and TH (detected via Alexa 633) were imaged with an oil immersion 60 x objective and 1.5 x optical zoom using an Olympus FV1000 confocal microscope. For quantification, images were processed for background subtraction in Fiji using the ‘rolling ball’ algorithm with a radius of 1 µm for each channel. Each background subtracted image was analyzed in a custom MATLAB program  and shape thresholds (ratio of x to y axis <1.5) were applied for object detection. These threshold settings were identical for each image and allowed for unbiased and automated detection of Bassoon-positive (Bassoon+), TH-positive (TH+) and Synaptotagmin-1-positive (Syt-1+) ROIs in each image, with a total of 300–600 synaptosome objects detected per image. These ROIs were then used to generate the Bassoon+TH+, Bassoon-TH+, Bassoon+TH- ROIs displayed in + ROIs with the various Bassoon/TH ROIs was quantified and a 20–100% overlap criterion was applied to define Synaptotagmin-1 positivity for the various Bassoon/TH ROIs. Sample images in Single optical sections of synaptosomes  of anesthetized male and female mice using stereotaxy (73–103 days old). Striatal dialysates were collected every 15 min and the concentration of dopamine was measured using an HPLC  connected to an electrochemical detector (Eicom). The data during the first 75 min were not plotted because during this time window dopamine levels stabilize after surgery. Average dopamine levels from the 76th - 120th min of Syt-1 control mice were used to normalize all dopamine values. 10 µM TTX dissolved in ACSF was applied using reverse dialysis starting at 121 min to inhibit firing of dopamine axons as described before ; Pablo Castillo (Reviewer #3).Thank you for submitting your article "Synaptotagmin-1 is the CaeLife papers without additional data. Thus the revisions requested below only address clarity and presentation.The reviewers have discussed the reviews with one another, and the Editors drafted this decision to help you prepare a revised submission in which we ask for some minor revisions. We would like to draw your attention to changes in our revision policy that we have made in response to COVID-19 (https://elifesciences.org/articles/57162). Specifically, we ask editors to accept without delay manuscripts, like yours, that they judge can stand as Summary:2+ sensor for fast dopamine release in striatal neurons. Despite the obvious importance of dopamine, the molecular machinery that governs release of dopamine from nerve terminals remains enigmatic. The authors used interdisciplinary techniques including unequivocal mouse genetics, elegant electrophysiology, optogenetics, carbon fiber amperometry, high resolution imaging and in vivo microdialysis to show that synaptotagmin-1 acts as a Ca2+ sensor for rapid exocytosis of dopaminergic synaptic vesicles in striatal neurons. Syt-1 deletion completely abolished fast synchronous release of dopamine. These terminals were capable of Syt-1-independent asynchronous release that accounted for roughly two thirds of extracellular dopamine levels. Overall, this is a straightforward study with convincing evidence establishing Syt-1 as the main Ca2+ sensor for fast dopamine release in striatal neurons. All three reviewers were enthusiastic about this work and had only the following minor concerns. The editors will assess your response at the time of the revision.Banerjee et al. aimed to determine the Ca1) How does Syt-1 deletion affect miniature synaptic responses in these neurons?2) How does external Ca concentration affect asynchronous dopamine release? Recordings in the Syt-1 DAT- cKO with dopamine re-uptake blocker provides an unique opportunity to explore this a bit further.2+ sensor for asynchronous dopamine release?3) The authors mention that Syt-7 levels are also high in midbrain dopaminergic neurons. Could Syt-7 be the Ca-mediated depolarization. See also point 9.4) Puffing of 100mM KCl in Figure 2 F-H. The authors showed that puffing high concentrations of KCl induced similar amount of dopamine release in dopamine neuronal conditional Synaptotagmin -1 knockout and in control. Can the authors elaborate on the nature of dopamine release induced by high KCl2+, was extracellular Mg2+ appropriately adjusted?5) For each manipulation of extracellular Ca6) Regarding the Syt-1 cKO DA slices, the traces in Figures 1H and 3A and the normalization in 1I indicate a slow accumulation of dopamine over time as a result of optical stimulation. The Syt-1 cKO DA dopamine traces and normalization yielded from electrical stimulation  show a much more stable dopamine level closer to 0. What is the potential explanation for this discrepancy?7) Figure 3C: The legend indicates that these measurements approximate total dopamine accumulated by integrating the dopamine curves between 0 and 2.9 s following the 1st stimulus. It is therefore unclear why the baseline DA level in Syt-1 cKO DA slices is exactly at 0 when the traces in Figures 1H and 3A, as well as the normalization in 1I, show a slight accumulation of dopamine over time in these slices.8) Figure 3G: In the pre-TTX condition, the average dopamine level in Syt-1 cKO DA animals is significantly smaller than that in Syt-1 control animals at only 1 of 3 time points. Can the author provide an explanation?-induced release calcium-dependent?9) The authors' explanation for how KCl but not ChR2 is able to cause DA release in Syt-1 cKO DA slices is not clear. Is this high KCl10) The third paragraph of the main section seems to have been misplaced. The information at the start of the paragraph would help clarify rationale if presented sooner. All three reviewers were enthusiastic about this work and had only the following minor concerns. The editors will assess your response at the time of the revision.1) How does Syt-1 deletion affect miniature synaptic responses in these neurons?2+ sensor often increases miniature release . Second, and as we discuss in the paper, there appears to be a significant amount of extracellular dopamine in the extracellular space even after removal of action-potential triggered dopamine release. This is true for knockouts for synaptotagmin, for knockouts of RIM, and for wild type mice after reverse dialysis of TTX . In microdialysis experiments in the striatum, however, there is no robust increase in extracellular dopamine in Syt1 cKODA mice after TTX, suggesting that miniature release is not enhanced, or that dopamine clearance is efficient enough to counteract such an increase. Unfortunately, there is currently no reliable direct measurement for miniature dopamine release in the striatum. Notably, in the midbrain, individual spontaneous events can be detected via measuring GIRK-IPSCs  and these events are unaffected after knockout of RIM .This is an important question in at least two ways. First, it has been shown that for conventional synaptic vesicle exocytosis, knockout of the fast CaDA leads to increased spontaneous dopamine release in the striatum. But the observation that extracellular dopamine levels after TTX are not strongly increased suggests that there is no dramatic enhancement of miniature dopamine release, or that an enhancement is counteracted by dopamine clearance.”We have revised the manuscript to better explain what is known about miniature release in the dopamine system. We now state in the manuscript: “It is not known whether synaptotagmin-1 knockout affects spontaneous dopamine release, but literature from conventional synapses establishes that synaptotagmin-1 knockout strongly enhances miniature synaptic vesicle release . Hence, it is possible that Syt-1 cKO2) How does external Ca concentration affect asynchronous dopamine release? Recordings in the Syt-1 DAT- cKO with dopamine re-uptake blocker provides an unique opportunity to explore this a bit further.2+ ]ex dependence of asynchronous dopamine release, and realize that we did not properly introduce the topic of asynchronous release in the text. While there is a large body of literature that establishes complex but positive correlations between [Ca2+ ]ex and asynchronous release , we currently do not know what the [Ca2+ ]ex dependence of asynchronous dopamine release is. The strong prediction from conventional synapses is that asynchronous dopamine release is positively correlated with [Ca2+ ]ex, but characterization beyond this point will require better measurements than accumulation during stimulus trains upon DAT blockade. We hope that recent developments in measurements of dopamine release will allow in the future to detect individual secretory events and their timing relative to action potential firing during trains. To better introduce asynchronous release, we added the following statement: “These additional sensors may for example mediate asynchronous release, a form of vesicular exocytosis at synapses that is triggered with a longer, variable delay in response to action potentials and Ca2+ entry .”We thank the reviewers for bringing up the ex .”Yes, extracellular Mg2+]ex before without adjusting Mg2+ , and that the increase in dopamine release was similar to what we report here.We note that we have done experiments increasing [Ca6) Regarding the Syt-1 cKO DA slices, the traces in Figures 1H and 3A and the normalization in 1I indicate a slow accumulation of dopamine over time as a result of optical stimulation. The Syt-1 cKO DA dopamine traces and normalization yielded from electrical stimulation  show a much more stable dopamine level closer to 0. What is the potential explanation for this discrepancy?The key difference between the two experiments is that in the main figures , optogenetic stimulation was used while in the figure supplement  electrical stimulation was applied. Optogenetic stimulation only recruits dopamine axons and leads to depression. In contrast, electrical stimulation recruits dopamine axons and cholinergic interneurons, and stimulating cholinergic interneurons strongly enhances depression . Hence, the discrepancy is explained by recruitment of the cholinergic mechanism and is consistent with previous papers published on this topic. We now state in the figure legend of Figure 1—figure supplement 4: “Electrical stimulation activates dopamine fibers and cholinergic interneurons, and cholinergic innervation of dopamine axons accounts for as much as ~90% of the extracellular dopamine detected upon electrical stimulation and leads to enhanced depression of dopamine release during stimulus trains .”7) Figure 3C: The legend indicates that these measurements approximate total dopamine accumulated by integrating the dopamine curves between 0 and 2.9 s following the 1st stimulus. It is therefore unclear why the baseline DA level in Syt-1 cKO DA slices is exactly at 0 when the traces in Figures 1H and 3A, as well as the normalization in 1I, show a slight accumulation of dopamine over time in these slices.DA was not exactly zero, but the scale of the y-axis was such that it was difficult to see this. We have now split up the graph in Figure 3C such that Syt1 control and Syt1 cKODA values are shown on separate scales and the low amount of accumulation at baseline can be visualized. We thank the reviewers for pointing this out.We thank the reviewers for bringing this up and would like to note that the baseline DA level in Syt1 cKO8) Figure 3G: In the pre-TTX condition, the average dopamine level in Syt-1 cKO DA animals is significantly smaller than that in Syt-1 control animals at only 1 of 3 time points. Can the author provide an explanation?DA in Figure 3G is surprisingly mild given the strong reduction in synchronous release. Because the pre-TTX effect of Syt1 cKODA is mild and there is some variability in microdialysis measurements, it is difficult to detect. This is particularly striking because in mutants that lack RIM in dopamine neurons in which action potential triggered release is also absent, pre-TTX dopamine levels in microdialysis are very strongly reduced .In our view, the pre-TTX effect of Syt1 cKODA…”.We describe the effect of synaptotagmin-1 ablation on pre-TTX dopamine in vivo as follows : “Remarkably, extracellular dopamine levels before TTX reverse dialysis were only mildly reduced in Syt-1 cKODA mice, and changes in the clearance of dopamine could also contribute. We also note that there is a good match across different mutants between dopamine in the microdialysate and KCl depolarization induced release: in Syt1 cKODA animals, amperometric currents in response to action potentials are strongly reduced, but KCl depolarization and in vivo microdialysis reports significant extracellular dopamine. This is different from dopamine neuron RIM knockouts (RIM cKODA), in which KCl triggered release is abolished and pre-TTX extracellular dopamine in microdialysis is strongly reduced . To summarize these effects, we now state the following: “These data suggest that three modes of dopamine release exist: synchronous and asynchronous release in response to action potentials, and action potential independent release that is likely mediated by spontaneous exocytotic events. Remarkably, each component appears to contribute significantly to the extracellular dopamine measured by microdialysis in anesthetized mice, suggesting that asynchronous and action potential-independent release may be prominent.”While we don’t have a definitive answer as to why this is the case, we think it is most likely that in vivo, asynchronous release contributes robustly to extracellular dopamine because dopamine levels in microdialysis drop significantly after action potential blockade with TTX in Syt-1 cKOWe hope that these statements, together with the explanations above, address this concern.-induced release calcium-dependent?9) The authors' explanation for how KCl but not ChR2 is able to cause DA release in Syt-1 cKO DA slices is not clear. Is this high KClWe thank the reviewers to bring up this important point, and it relates to point 4 as well. This point has two aspects: (1) how channelrhodopsin triggers release, and (2) how KCl triggers release.2+ enters through channelrhodopsin directly under some circumstances, this is not the case here in a way that Ca2+ is sufficient to trigger dopamine release. The experiments in Syt1 cKODA animals are performed with oChiEF, not ChR2, and we have directly tested whether sodium channels are necessary for this form of release. We found that TTX entirely blocks oChiEF induced release . Hence, this method triggers release by evoking action potentials in dopamine axons. We now state: “In these mice, we expressed oChiEF-citrine, a fast channelrhodopsin, selectively in dopamine neurons using AAVs  to optogenetically evoke dopamine release through triggering of axonal action potentials .”1) While it is possible that CaDA mice, we conclude that KCl triggers vesicular exocytosis of dopamine through strong depolarization of dopamine axons.2) As outlined above, KCl triggers release most likely through at least two mechanisms, massive depolarization of (i) dopamine axons and (ii) of cholinergic interneurons, which in turn release acetylcholine and then trigger dopamine release through an unknown mechanism after activation of dopamine axonal nAChRs. Notably, this form of dopamine release requires the presence of vesicular release machinery, for example RIM, and hence we concluded in a previously published study that it is vesicular . Because release triggered through nAChRs activation  is abolished in Syt1 cKOWe now specifically describe the mechanisms of KCl mediated release: “Local puffing of KCl onto brain slices causes a strong depolarization of dopamine axons and surrounding neurons, for example cholinergic interneurons, which triggers massive dopamine release that requires the active zone protein RIM in dopamine axons .”2+ for this form of release would be to remove extracellular Ca2+ entirely. For optogenetic experiments, we have tried to fully remove extracellular Ca2+ from striatal slices, but have found that this is challenging and we do not have good experiments to evaluate the requirement of extracellular Ca2+ for KCl-triggered dopamine release. Hence, although there is a long-term body of literature to suggest that KCl triggers release through depolarization-dependent opening of voltage gated Ca2+ channels followed by flooding of the nerve terminal with Ca2+, we do not have the capability to prove this at this point in striatal brain slices. To account for this in the manuscript, we now state: “While the exact mechanism of KCl depolarization induced dopamine release is not known, the data further suggest that depolarization-induced, likely massive Ca2+ entry may trigger vesicular dopamine release via one or multiple alternative Ca2+ sensors in the absence of Synaptotagmin-1.”The best experiment to directly test the requirement for extracellular Ca10) The third paragraph of the main section seems to have been misplaced. The information at the start of the paragraph would help clarify rationale if presented sooner.We thank the reviewers for this stylistic suggestion and have switched the paragraphs as suggested to present some of the rationale sooner.References78, 807–812.Gantz, S.C., Bunzow, J.R., and Williams, J.T. (2013). Spontaneous Inhibitory Synaptic Currents Mediated by a G Protein-Coupled Receptor. Neuron Life8.Robinson, B.G., Cai, X., Wang, J., Bunzow, J.R., Williams, J.T., and Kaeser, P.S. (2019). RIM is essential for stimulated but not spontaneous somatodendritic dopamine release in the midbrain. e


KMT2D and KDM6A, are responsible for KS1 and KS2, respectively.Kabuki syndrome (KS) is a rare congenital condition with cardinal manifestations of typical facial features, developmental delays, skeletal anomalies, abnormal dermatoglyphic presentations, and mild to moderate intellectual disability. Pathogenic variants in two epigenetic modifier genes, KMT2D (NM_003482.3 c.12165del p.(Glu4056Serfs*10)) that caused frameshift and premature termination. The mutation was de novo. According to the American College of Medical Genetics and Genomics (ACMG) guidelines, this variant is considered pathogenic. The patient was diagnosed with KS by molecular testing.A Chinese girl had persistent neonatal hypoglycemia and Dandy-Walker variant. Whole-exome sequencing identified a novel single nucleotide deletion in KMT2D was identified in a KS patients with hypoglycemia and Dandy-Walker variant in the neonatal stage. A molecular test was conducted to diagnose KS at an early stage.A single novel mutation in  KMT2D and KDM6A are responsible for KS 1 (MIM: 147920) [KMT2D is located on chromosome 12. Pathogenic variants of KMT2D are found in approximately 75% of KS cases [KDM6A is located on chromosome X and is inherited as X-linked dominant. Pathogenic variants are found in only 3–5% of KS patients [Kabuki syndrome  is a rare condition with an incidence of approximately 1 in 32,000 people [ 147920) , respectKS is seldom diagnosed during the newborn period. First, the generally consistent features of KS, such as the typical facial features and persistent fetal finger pads, become increasingly obvious as age increases. Second, the typical facial features are less pronounced at the neonatal stage, resulting in many cases going unnoticed by doctors who are not familiar with KS. Here, we report the case of a young girl who experienced hypoglycemia and had Dandy-Walker variant; she was diagnosed with KS1 in the second month after birth by whole-exome sequencing. Dandy-Walker variant has rarely been reported in KS patients before.A young girl was the second child of a nonconsanguineous couple of Chinese ancestry. Her elder sister was normal, and her family history was unremarkable. The patient was naturally conceived when her mother was 36 years old. Nuchal translucency (NT) was 1.1 mm at the first-trimester screening. No apparent defect was observed on a routine second-trimester ultrasound. However, ultrasound examination revealed polyhydramnios at 30 and 37 weeks. The patient was born at 38 weeks gestation by cesarean delivery and had normal birth parameters (Supplementary Table KMT2D (NM_003482.3), which was predicted to cause coding sequence frameshift and premature translation termination (p.(Glu4056Serfs*10)). The variant was confirmed by Sanger sequencing and was tested for familial segregation demonstrating de novo occurrences  and sequenced on a MiSeq platform . The variants with an allele frequency (AF) > 1% in gnomAD felt out. The mutations that were previously reported, considered damaging  or absent in gnomAD were given priority. The gene set on the basis of Human Phenotype Ontology (HPO) HP: 0001943) was applied to narrow down the candidate gene list. By analyzing the sequencing data, we found a heterozygous single nucleotide deletion (c.12165del) in the 39th (of 54) exon of ces Fig. . None of3 was appKS is a congenital condition characterized by typical facial features, infantile hypotonia, mild to moderate intellectual disability, persistent fetal fingertip pads, and global developmental delay. The diagnosis is traditionally initially made according to the clinical features and then confirmed by molecular diagnosis. However, not all patients have typical clinical features, especially Chinese patients . TherefoHypoglycemia is not a cardinal manifestation of KS. Only 8–10% of patients with KS have hypoglycemia during the neonatal or infantile stage . The cauIt has been suggested that KS is correlated with brain anomalies. Various structural brain anomalies, including temporoparietal subarachnoid cyst , perisylKMT2D mutation spectrum includes nonsense mutations, frameshift mutations, missense mutations, splicing site mutations, gross deletions, gross duplications, and indels, among which more than 60% are nonsense mutations and frameshift mutations [The utations . Therefoutations , ClinVarutations . Accordiutations .In conclusion, this case study reports an early diagnosis of KS1 in a patient with the atypical phenotype, hypoglycemia and Dandy-Walker variant by genetic testing. These data broaden the mutation spectrum and add information about phenotypes relating to KS. A molecular diagnosis should be considered for patients suspected of having genetic disorders to discover and differentiate rare diseases.Additional file 1: Supplementary Table 1. Birth parameter. Supplementary Table 2. Blood gas result. Supplementary Table 3. Liver function test. Supplementary Table 4. The variants in genes regulating insulin secretion of coding region and splicing region(±10).Additional file 2. Supplementary Method.


This review outlines recent preclinical and clinical advances in molecular imaging of abdominal aortic aneurysms (AAA) with a focus on molecular magnetic resonance imaging (MRI) of the extracellular matrix (ECM). In addition, developments in pharmacologic treatment of AAA targeting the ECM will be discussed and results from animal studies will be contrasted with clinical trials. Abdominal aortic aneurysm (AAA) is an often fatal disease without non-invasive pharmacologic treatment options. The ECM, with collagen type I and elastin as major components, is the key structural component of the aortic wall and is recognized as a target tissue for both initiation and the progression of AAA. Molecular imaging allows in vivo measurement and characterization of biological processes at the cellular and molecular level and sets forth to visualize molecular abnormalities at an early stage of disease, facilitating novel diagnostic and therapeutic pathways. By providing surrogate criteria for the in vivo evaluation of the effects of pharmacological therapies, molecular imaging techniques targeting the ECM can facilitate pharmacological drug development. In addition, molecular targets can also be used in theranostic approaches that have the potential for timely diagnosis and concurrent medical therapy. Recent successes in preclinical studies suggest future opportunities for clinical translation. However, further clinical studies are needed to validate the most promising molecular targets for human application. Abdominal aortic aneurysm (AAA) is a fatal, but often asymptomatic disease with controversial treatment and insufficient prediction of complications ,2. The eIn clinical routine, morphological criteria for the determination of the aortic diameter on ultrasound, computed tomography (CT) and MRI have been used in accordance with guidelines as evaluation criteria for therapeutic intervention or conservative therapy. By contrast, molecular imaging enables an in vivo measurement and characterization of biological processes at cellular and molecular level and sets forth to visualize molecular abnormalities at an early stage of disease, facilitating novel diagnostic and also therapeutic pathways . MRI is In the context of AAA, development of pharmacological therapies is of high clinical relevance. It has been estimated that inhibiting the growth of an AAA by 40% could delay the occurrence of a rupture by possibly five years ,9. ConseIn each organ, the composition of the ECM has a different three-dimensional structure and a constant pattern of remodeling to regulate tissue homeostasis . Within Molecular MRI includes both targeted probes with selective binding to molecular targets and probes that accumulate passively within cells  . A majorIn vivo imaging of AAA wall inflammation and ECM remodeling has previously been achieved using ultrasmall superparamagnetic particles of iron oxide (USPIOs), elastin-specific MRI contrast agents (Gd-ESMA) as well as collagen- and fibrin-binding probes ,22,23,243RS study, USPIO enhancement showed no correlation with computed tomography angiogram (CTA) predictions of stress areas, so that localization of rupture-prone areas in the clinical setting has not yet been successful [In a recently published prospective multicenter cohort study (MA3RS) including 342 patients with abdominal aortic aneurysms, USPIO enhancement was associated with higher risk of aortic rupture or repair, reduced event-free survival from aneurysm rupture or repair, and aneurysm expansion, although it was not independent of clinical risk factors and thus had limited additional value beyond current clinical risk prediction . In a successful .A further crucial process in AAA development, related in part to inflammation, is remodeling of the ECM. Dysfunctional ECM remodeling may reduce wall stability and promote AAA formation. Gd-ESMA is an elastin-specific MRI probe that showed high potential in quantitative imaging of vascular diseases . By specAnother protein that may be increased in AAA due to dysfunctional ECM remodeling is tropoelastin, a monomeric precursor of cross-linked elastin . A recenMMPs are also associated with changes in the ECM and the development of AAA. P947 is a gadolinium (DOTA)-binding molecular probe that specifically binds to MMPs, particularly MMP-2 and MMP-9. In AAA-induced wistar rats, P947 MRI images showed good colocalization of the sample with MMPs in wall areas of inflammatory events .99mTc-labeled homolog, RP805 predicted vessel expansion and rupture probability in Ang-II-induced murine AAA [99mTc-labelled pan-MMP inhibitor RYM1 and demonstrated a higher uptake of the tracer in AAA compared to nondilated aorta [MMP tracers have also been developed for nuclear imaging. The rine AAA . Anothered aorta . In addied aorta .Yao et al. investigated a smart activatable MRI nanoprobe to target MMP in early-stage AAA in a mouse model and found that their probe allowed for the detection of MMP activity within the aneurysmal wall, thus representing a potential noninvasive method to predict the risk of rupture in AAA [A recent study by e in AAA .vβ3 is upregulated in proliferating endothelial cells, VSMC and macrophages [99mTc-cyclic RGD tracer for microSPECT-CT displayed increased uptake in murine carotid aneurysms and a correlation with inflammatory activity [64Cu-DOTA-ECL1i [64Cu-DOTA-ECL1i showed significantly increased uptake compared with sham controls and compared with aneurysms that did not rupture during progression [More consistent results were achieved in preclinical studies on integrin-targeted tracers. Integrin αrophages ,35. NC10activity . EnglishTA-ECL1i . CCR2 isgression .Experimental targets for pharmaceutical AAA stabilization, that target the ECM, are thrombospondin inhibitors , cysteinIn humans, elevated thrombospondin-1 (TSP-1) was associated with MMP activitaion, ECM degradation as well as tissue infiltration . CysteinAneurysmal collagen and elastin degradation is caused by a number of endopeptidases called MMPs. MMPs belong to a family of enzymes whose main function is the degradation of ECM components and the disruption of tissue organization ,51. MMP Being among the most abundant elastolytic proteinases produced by human tissue and aneurysm-infiltrating macrophages, MMP-9 has gained particular interest. In a molecular MRI study, Bazeli et al. reported the potential value of an MMP-targeted probe (P947) in rats. P947 was shown to co-localize with markers of inflammation and MMP activity in the area of expanding AAA .99mTc-AGA-2 in murine models, for which they demonstrated specific binding to MMP-12 through ex vivo competition, and found it to be significantly increased in AAA compared to healthy aortae [MMP-12 is the most upregulated MMP in AAA and may therefore be particularly valuable for prediction of AAA progression and rupture risk. Gona et al. recently investigated the MMP-12-inhibitor y aortae .Katsuki et al. recently investigated the use of pitavastatin in a mouse model of AAA and found that it could inhibit AAA formation, being associated with reduced macrophage accumulation, MMP activity and elastin degradation . However3CT)), reporting that doxycycline (200 mg/day) did not reduce AAA growth at 2 years as compared to placebo in patients with small infrarenal AAA [3CT trial corresponded to circulating levels of doxycycline that were required to reduce AAA growth in preclinical studies with mice [The tetracycline antibiotic doxycycline has been studied in the context of AAA for many years. In a series of earlier preclinical studies doxycycline effectively interfered with aneurysm formation or growth ,64. Prevenal AAA . While iith mice . The proPentagalloyl glucose (PGG) is a polyphenolic tannin. In a mouse model of AAA, twice IV application of nanoparticle-loaded PGG (EL-PGG-NPs) showed a reduction of MMP-9, MMP-2 and of macrophages in the vascular media and restored the elastic lamina . FucoidaADAMs and ADAMs with a thrombospondin domain (ADAM-TS) are zinc-dependent endopeptidases and belong to the proteolytic enzyme family, involved in ECM degradation and closely related to other MMPs . They boMicroRNAs (miRNA) are small, 19–24 nucleotide, highly conserved non-coding RNAs that are involved in gene regulation by binding to the 3′ untranslated region of mRNA, thereby inhibiting translation. Through this mechanism, miRNAs are involved in diverse processes of cellular tumorigenesis, apoptosis, and differentiation . In addiWang et al. used ultrasound microbubbles coupled with a VCAM-1-targeted single-chain antibody (scFvmVCAM-1) and a miRNA-126mimic (M126) as carriers, enabling a theranostic approach (TargMB-M126) with simultaneous molecular imaging and targeted therapy of AAA in a mouse model of AAA [Another potential therapeutic target is miRNA-126 with a capability to downregulate vascular cell adhesion molecule-1 (VCAM1) and thereby reduce leukocyte adhesion with a subsequent anti-inflammatory effect . Wang etl of AAA   indicated that anti-miRNA29b treatment was a powerful therapeutic option to limit AAA progression . In addiThe degradation of ECM components is induced in part by cytokines secreted by inflammatory and mesenchymal cells. In this context, macrophages in particular act as important releasers of a variety of proinflammatory cytokines, including tumor necrosis factor α (TNF-α), interleukin (IL)-1β, IL-6, IL-12/IL-23 . Apart fIncreased production of IL-6 causes degradation of the ECM with an increased activity of MMP-9 and a consecutive aortic dilatation. IL-6 deficiency was previously demonstrated to improve AAA pathology by partially preserving ECM structure through a reduction of MMP-9 expression, whereas it could neither prevent rupture nor increase survival .Inhibition of IL-12 and IL-23 has been shown to inhibit macrophage expansion and, in particular, to reduce the expression of macrophage-associated inflammatory mediators and the activity of MMPs . TherefoInterleukin-1β functions as a gatekeeper of inflammation and contributes substantially to the progressive destruction of aortic ECM proteins during the development of AAA. Disruption of the inflammatory pathway by neutralizing IL-1β represents a promising new therapeutic target for AAA therapies . In a moIn addition, they suggested that molecular imaging allowed for differentiation between responders and non-responders early after initiation of therapy .n = 65), but the trial was terminated afterwards for reasons of futility as no difference in aortic diameter expansion could be seen between canakinumab- and placebo-treated patients (both groups: 2.5 mm/y) [In humans, effect of IL-1β neutralization through monthly subcutaneous canakinumab for twelve months was evaluated (.5 mm/y) .Over the past 30 years, a variety of promising gadolinium-based molecular tracers have been developed and their utility confirmed in proof-of-concept animal studies. Nevertheless, only non-targeted contrast agents have made it to clinical approval for use in humans. A challenge in clinical translation is that toxicological testing is time-consuming and costly, requiring substantial financial resources. This contradicts the expected revenues, which are lower for molecular tracers than for clinically proven nonspecific contrast agents due to their limited application in specific settings. Nonetheless, these markers play a prominent role especially in preclinical research and, if validated, can non-invasively image new therapeutic approaches, for example targeting ECM remodeling. In this context, the elastin-specific MRI probe Gd-ESMA and tropoelastin-specific MRI probe Gd-TESMA appear promising, as they can specifically detect the effect of therapeutic strategies affecting the elastin content in the aortic wall via their different pathways. Another novel approach are multitarget probes, which could allow for concurrent imaging of different pathophysiological processes at the molecular level. Molecular agents may also be used within theranostic approaches with the potential for timely diagnosis and concurrent medical therapy. Here, the use of miRNA-based targets is a novel and promising method.Currently, there is no established pharmacologic therapy for the treatment of AAA. The apparent discordance between successes in preclinical studies and partly disappoint results in clinical trials suggests an incomplete understanding of the various pathological processes involved in the development of AAAs and points to possible inadequacies in animal models of the disease. To increase future therapeutic strategies, molecular imaging markers that can specifically visualize and quantify targets of pharmacological agents in ECM in a non-invasive manner hold promise for a better reflection of the pathophysiological processes behind the disease and during therapy. In addition, recent preclinical approaches in large animal models represent a promising option for evaluating therapeutic strategies and create better options for multiparametric functional imaging, which seems to be limited in parts by the low volume in small animal models. With the increasing availability of image-based molecular biomarkers and promising pharmacotherapies, as well as their evaluation in large animal models of AAA and initial human clinical trials, there is reason to hope that molecular therapeutic approaches targeting ECM will enter clinical research in the coming years.