Best practices for user consultation in flow cytometry shared resource laboratories.

This "Best Practices in User Consultation" article is the result of a 2022 International Society for the Advancement of Cytometry (ISAC) membership survey that collected valuable insights from the shared research laboratory (SRL) community and of a group discussion at the CYTO 2022 workshop of the same name. One key takeaway is the importance of initiating a consultation at the outset of a flow cytometry project, particularly for trainees. This approach enables the improvement and standardization of every step, from planning experiments to interpreting data. This proactive approach effectively mitigates experimental bias and avoids superfluous trial and error, thereby conserving valuable time and resources. In addition to guidelines, the optimal approaches for user consultation specify communication channels, methods, and critical information, thereby establishing a structure for productive correspondence between SRL and users. This framework functions as an exemplar for establishing robust and autonomous collaborative relationships. User consultation adds value by providing researchers with the necessary information to conduct reproducible flow cytometry experiments that adhere to scientific rigor. By following the steps, instructions, and strategies outlined in these best practices, an SRL can readily tailor them to its own setting, establishing a personalized workflow and formalizing user consultation services. This article provides a pragmatic guide for improving the caliber and efficacy of flow cytometry research and aggregates the flow cytometry SRL community's collective knowledge regarding user consultation.

Contrasting neurofunctional correlates of face- and visuospatial-processing in children and adolescents with Williams syndrome: convergent results from four fMRI paradigms.

Understanding neurogenetic mechanisms underlying neuropsychiatric disorders such as schizophrenia and autism is complicated by their inherent clinical and genetic heterogeneity. Williams syndrome (WS), a rare neurodevelopmental condition in which both the genetic alteration (hemideletion of ~ twenty-six 7q11.23 genes) and the cognitive/behavioral profile are well-defined, offers an invaluable opportunity to delineate gene-brain-behavior relationships. People with WS are characterized by increased social drive, including particular interest in faces, together with hallmark difficulty in visuospatial processing. Prior work, primarily in adults with WS, has searched for neural correlates of these characteristics, with reports of altered fusiform gyrus function while viewing socioemotional stimuli such as faces, along with hypoactivation of the intraparietal sulcus during visuospatial processing. Here, we investigated neural function in children and adolescents with WS by using four separate fMRI paradigms, two that probe each of these two cognitive/behavioral domains. During the two visuospatial tasks, but not during the two face processing tasks, we found bilateral intraparietal sulcus hypoactivation in WS. In contrast, during both face processing tasks, but not during the visuospatial tasks, we found fusiform hyperactivation. These data not only demonstrate that previous findings in adults with WS are also present in childhood and adolescence, but also provide a clear example that genetic mechanisms can bias neural circuit function, thereby affecting behavioral traits.

Dopamine signaling enriched striatal gene set predicts striatal dopamine synthesis and physiological activity in vivo.

The polygenic architecture of schizophrenia implicates several molecular pathways involved in synaptic function. However, it is unclear how polygenic risk funnels through these pathways to translate into syndromic illness. Using tensor decomposition, we analyze gene co-expression in the caudate nucleus, hippocampus, and dorsolateral prefrontal cortex of post-mortem brain samples from 358 individuals. We identify a set of genes predominantly expressed in the caudate nucleus and associated with both clinical state and genetic risk for schizophrenia that shows dopaminergic selectivity. A higher polygenic risk score for schizophrenia parsed by this set of genes predicts greater dopamine synthesis in the striatum and greater striatal activation during reward anticipation. These results translate dopamine-linked genetic risk variation into in vivo neurochemical and hemodynamic phenotypes in the striatum that have long been implicated in the pathophysiology of schizophrenia.

Altered pubertal timing in 7q11.23 copy number variations and associated genetic mechanisms.

Pubertal timing, including age at menarche (AAM), is a heritable trait linked to lifetime health outcomes. Here, we investigate genetic mechanisms underlying AAM by combining genome-wide association study (GWAS) data with investigations of two rare genetic conditions clinically associated with altered AAM: Williams syndrome (WS), a 7q11.23 hemideletion characterized by early puberty; and duplication of the same genes (7q11.23 Duplication syndrome [Dup7]) characterized by delayed puberty. First, we confirm that AAM-derived polygenic scores in typically developing children (TD) explain a modest amount of variance in AAM (R2 = 0.09; p = 0.04). Next, we demonstrate that 7q11.23 copy number impacts AAM (WS < TD < Dup7; p = 1.2x10-8, η2 = 0.45) and pituitary volume (WS < TD < Dup7; p = 3x10-5, ηp2 = 0.2) with greater effect sizes. Finally, we relate an AAM-GWAS signal in 7q11.23 to altered expression in postmortem brains of STAG3L2 (p = 1.7x10-17), a gene we also find differentially expressed with 7q11.23 copy number (p = 0.03). Collectively, these data explicate the role of 7q11.23 in pubertal onset, with STAG3L2 and pituitary development as potential mediators.

Syntaxin1A overexpression and pain insensitivity in individuals with 7q11.23 duplication syndrome.

Genetic modifications leading to pain insensitivity phenotypes, while rare, provide invaluable insights into the molecular biology of pain and reveal targets for analgesic drugs. Pain insensitivity typically results from Mendelian loss-of-function mutations in genes expressed in nociceptive (pain-sensing) dorsal root ganglion (DRG) neurons that connect the body to the spinal cord. We document a pain insensitivity mechanism arising from gene overexpression in individuals with the rare 7q11.23 duplication syndrome (Dup7), who have 3 copies of the approximately 1.5-megabase Williams syndrome (WS) critical region. Based on parental accounts and pain ratings, people with Dup7, mainly children in this study, are pain insensitive following serious injury to skin, bones, teeth, or viscera. In contrast, diploid siblings (2 copies of the WS critical region) and individuals with WS (1 copy) show standard reactions to painful events. A converging series of human assessments and cross-species cell biological and transcriptomic studies identified 1 likely candidate in the WS critical region, STX1A, as underlying the pain insensitivity phenotype. STX1A codes for the synaptic vesicle fusion protein syntaxin1A. Excess syntaxin1A was demonstrated to compromise neuropeptide exocytosis from nociceptive DRG neurons. Taken together, these data indicate a mechanism for producing "genetic analgesia" in Dup7 and offer previously untargeted routes to pain control.

Guidelines for establishing a cytometry laboratory.

The purpose of this document is to provide guidance for establishing and maintaining growth and development of flow cytometry shared resource laboratories. While the best practices offered in this manuscript are not intended to be universal or exhaustive, they do outline key goals that should be prioritized to achieve operational excellence and meet the needs of the scientific community. Additionally, this document provides information on available technologies and software relevant to shared resource laboratories. This manuscript builds on the work of Barsky et al. 2016 published in Cytometry Part A and incorporates recent advancements in cytometric technology. A flow cytometer is a specialized piece of technology that require special care and consideration in its housing and operations. As with any scientific equipment, a thorough evaluation of the location, space requirements, auxiliary resources, and support is crucial for successful operation. This comprehensive resource has been written by past and present members of the International Society for Advancement of Cytometry (ISAC) Shared Resource Laboratory (SRL) Emerging Leaders Program https://isac-net.org/general/custom.asp?page=SRL-Emerging-Leaders with extensive expertise in managing flow cytometry SRLs from around the world in different settings including academia and industry. It is intended to assist in establishing a new flow cytometry SRL, re-purposing an existing space into such a facility, or adding a flow cytometer to an individual lab in academia or industry. This resource reviews the available cytometry technologies, the operational requirements, and best practices in SRL staffing and management.

PET-measured human dopamine synthesis capacity and receptor availability predict trading rewards and time-costs during foraging.

Foraging behavior requires weighing costs of time to decide when to leave one reward patch to search for another. Computational and animal studies suggest that striatal dopamine is key to this process; however, the specific role of dopamine in foraging behavior in humans is not well characterized. We use positron emission tomography (PET) imaging to directly measure dopamine synthesis capacity and D1 and D2/3 receptor availability in 57 healthy adults who complete a computerized foraging task. Using voxelwise data and principal component analysis to identify patterns of variation across PET measures, we show that striatal D1 and D2/3 receptor availability and a pattern of mesolimbic and anterior cingulate cortex dopamine function are important for adjusting the threshold for leaving a patch to explore, with specific sensitivity to changes in travel time. These findings suggest a key role for dopamine in trading reward benefits against temporal costs to modulate behavioral adaptions to changes in the reward environment critical for foraging.

Dorsal visual stream and LIMK1: hemideletion, haplotype, and enduring effects in children with Williams syndrome.

BACKGROUND: Williams syndrome (WS), a rare neurodevelopmental disorder caused by hemizygous deletion of ~ 25 genes from chromosomal band 7q11.23, affords an exceptional opportunity to study associations between a well-delineated genetic abnormality and a well-characterized neurobehavioral profile. Clinically, WS is typified by increased social drive (often termed "hypersociability") and severe visuospatial construction deficits. Previous studies have linked visuospatial problems in WS with alterations in the dorsal visual processing stream. We investigated the impacts of hemideletion and haplotype variation of LIMK1, a gene hemideleted in WS and linked to neuronal maturation and migration, on the structure and function of the dorsal stream, specifically the intraparietal sulcus (IPS), a region known to be altered in adults with WS. METHODS: We tested for IPS structural and functional changes using longitudinal MRI in a developing cohort of children with WS (76 visits from 33 participants, compared to 280 visits from 94 typically developing age- and sex-matched participants) over the age range of 5-22. We also performed MRI studies of 12 individuals with rare, shorter hemideletions at 7q11.23, all of which included LIMK1. Finally, we tested for effects of LIMK1 variation on IPS structure and imputed LIMK1 expression in two independent cohorts of healthy individuals from the general population. RESULTS: IPS structural (p < 10-4 FDR corrected) and functional (p < .05 FDR corrected) anomalies previously reported in adults were confirmed in children with WS, and, consistent with an enduring genetic mechanism, were stable from early childhood into adulthood. In the short hemideletion cohort, IPS deficits similar to those in WS were found, although effect sizes were smaller than those found in WS for both structural and functional findings. Finally, in each of the two general population cohorts stratified by LIMK1 haplotype, IPS gray matter volume (pdiscovery < 0.05 SVC, preplication = 0.0015) and imputed LIMK1 expression (pdiscovery = 10-15, preplication = 10-23) varied according to LIMK1 haplotype. CONCLUSIONS: This work offers insight into neurobiological and genetic mechanisms responsible for the WS phenotype and also more generally provides a striking example of the mechanisms by which genetic variation, acting by means of molecular effects on a neural intermediary, can influence human cognition and, in some cases, lead to neurocognitive disorders.

Clinical correlation but no elevation of striatal dopamine synthesis capacity in two independent cohorts of medication-free individuals with schizophrenia.

Dysregulation of dopamine systems has been considered a foundational driver of pathophysiological processes in schizophrenia, an illness characterized by diverse domains of symptomatology. Prior work observing elevated presynaptic dopamine synthesis capacity in some patient groups has not always identified consistent symptom correlates, and studies of affected individuals in medication-free states have been challenging to obtain. Here we report on two separate cohorts of individuals with schizophrenia spectrum illness who underwent blinded medication withdrawal and medication-free neuroimaging with [18F]-FDOPA PET to assess striatal dopamine synthesis capacity. Consistently in both cohorts, we found no significant differences between patient and matched, healthy comparison groups; however, we did identify and replicate robust inverse relationships between negative symptom severity and tracer-specific uptake widely throughout the striatum: [18F]-FDOPA specific uptake was lower in patients with a greater preponderance of negative symptoms. Complementary voxel-wise and region of interest analyses, both with and without partial volume correction, yielded consistent results. These data suggest that for some individuals, striatal hyperdopaminergia may not be a defining or enduring feature of primary psychotic illness. However, clinical differences across individuals may be significantly linked to variability in striatal dopaminergic tone. These findings call for further experimentation aimed at parsing the heterogeneity of dopaminergic systems function in schizophrenia.

Comparison of Transcranial Sonography and [18 F]-Fluorodopa PET Imaging in GBA1 Mutation Carriers.

BACKGROUND: Mutations in GBA1 are a common genetic risk factor for parkinsonism; however, penetrance is incomplete, and biomarkers of future progression to parkinsonism are needed. Both nigral sonography and striatal [18 F]-FDOPA PET assay dopamine system health, but their utility and coherence in this context are unclear. OBJECTIVE: The aim of this study is to evaluate the utility and coherence of these modalities in GBA1-associated parkinsonism. METHODS: A total of 34 patients with GBA1 mutations (7 with parkinsonism) underwent both transcranial studies for substantia nigra echogenicity and [18 F]-FDOPA PET to determine striatal tracer-specific uptake (Ki ). RESULTS: Larger nigral echogenic areas and reduced striatal Ki were exclusively observed in parkinsonian patients. Sonographic and PET measurements showed strong inverse correlations but only in individuals with clinical parkinsonism. CONCLUSIONS: Close correspondence between nigral echogenicity and striatal presynaptic dopamine synthesis capacity observed only in GBA1 carriers with parkinsonism provides validation that these two modalities may conjointly capture aspects of the biology underlying clinical parkinsonism but raises questions about their utility as predictive tools in at-risk subjects. © 2022 International Parkinson and Movement Disorder Society.

Antipsychotic medication-mediated cognitive change in schizophrenia and polygenic score for cognitive ability.

Cognitive symptoms of schizophrenia are reported to be minimally responsive to treatment with antipsychotic medications, though variability exists and many prior studies have significant confounds. Here, we examined the response of cognitive symptoms to antipsychotic medications in 71 inpatients with schizophrenia on and off antipsychotic medications in a blinded, placebo-controlled, cross-over study design. Patients received either antipsychotic medication monotherapy or placebo for 4-6 weeks before switching conditions. Neuropsychological testing, including working memory, intelligence, episodic memory, and verbal fluency tests, was administered during each condition. Additionally, we assessed whether polygenic scores for cognitive ability (PGScog) related to variability in antipsychotic medication-induced changes in cognitive performance. Overall, significant changes in cognition were not observed in response to medications (p's > 0.05) except for in episodic memory (p = 0.01), which showed a medication-related improvement. Some antipsychotic medication-related cognitive changes were associated with genetic predisposition to cognitive ability: PGScog showed positive correlations with medication-induced improvements in verbal list learning (p = 0.02) and category fluency (p = 0.03). Our primary results reinforce the notion that in general, cognitive measures are minimally responsive to antipsychotic medication. However, PGScog results suggest that genetic variation may influence the ability of current treatments to affect cognitive change within this patient population. This study underscores the need for development of novel treatment options specifically targeting cognitive symptoms as well as the importance of genetic variability in treatment response for patients with schizophrenia.

Neanderthal-derived genetic variation in living humans relates to schizophrenia diagnosis, to psychotic symptom severity, and to dopamine synthesis.

Schizophrenia has been hypothesized to be a human-specific condition, but experimental approaches to testing this idea have been limited. Because Neanderthals, our closest evolutionary relatives, interbred with modern humans prior to their disappearance from the fossil record, leaving a residual echo that survives in our DNA today, we leveraged new discoveries about ancient hominid DNA to explore this hypothesis in living people in three converging ways. First, in four independent case-control datasets totaling 9,362 individuals, individuals with schizophrenia had less Neanderthal-derived genetic variation than controls (p = .044). Second, in 49 unmedicated inpatients with schizophrenia, having more Neanderthal admixture predicted less severe positive symptoms (p = .046). Finally, using 18 F-fluorodopa PET scanning in 172 healthy individuals, having greater Neanderthal introgression was significantly associated with lower dopamine synthesis capacity in the striatum and pons (p's < 2 × 10-5 ), which is fundamentally important in the pathophysiology and treatment of psychosis. These results may help to elucidate the evolutionary history of a devastating neuropsychiatric disease by supporting the notion of schizophrenia as a human-specific condition. Additionally, the relationship between Neanderthal admixture and dopamine function suggests a potential mechanism whereby Neanderthal admixture may have affected our gene pool to alter schizophrenia risk and/or course.

Beyond linearity in neuroimaging: Capturing nonlinear relationships with application to longitudinal studies.

The ubiquitous adoption of linearity for quantitative predictors in statistical modeling is likely attributable to its advantages of straightforward interpretation and computational feasibility. The linearity assumption may be a reasonable approximation especially when the variable is confined within a narrow range, but it can be problematic when the variable's effect is non-monotonic or complex. Furthermore, visualization and model assessment of a linear fit are usually omitted because of challenges at the whole brain level in neuroimaging. By adopting a principle of learning from the data in the presence of uncertainty to resolve the problematic aspects of conventional polynomial fitting, we introduce a flexible and adaptive approach of multilevel smoothing splines (MSS) to capture any nonlinearity of a quantitative predictor for population-level neuroimaging data analysis. With no prior knowledge regarding the underlying relationship other than a parsimonious assumption about the extent of smoothness (e.g., no sharp corners), we express the unknown relationship with a sufficient number of smoothing splines and use the data to adaptively determine the specifics of the nonlinearity. In addition to introducing the theoretical framework of MSS as an efficient approach with a counterbalance between flexibility and stability, we strive to (a) lay out the specific schemes for population-level nonlinear analyses that may involve task (e.g., contrasting conditions) and subject-grouping (e.g., patients vs controls) factors; (b) provide modeling accommodations to adaptively reveal, estimate and compare any nonlinear effects of a predictor across the brain, or to more accurately account for the effects (including nonlinear effects) of a quantitative confound; (c) offer the associated program 3dMSS to the neuroimaging community for whole-brain voxel-wise analysis as part of the AFNI suite; and (d) demonstrate the modeling approach and visualization processes with a longitudinal dataset of structural MRI scans.

The NIMH Intramural Longitudinal Study of the Endocrine and Neurobiological Events Accompanying Puberty: Protocol and rationale for methods and measures.

Delineating the relationship between human neurodevelopment and the maturation of the hypothalamic-pituitary-gonadal (HPG) axis during puberty is critical for investigating the increase in vulnerability to neuropsychiatric disorders that is well documented during this period. Preclinical research demonstrates a clear association between gonadal production of sex steroids and neurodevelopment; however, identifying similar associations in humans has been complicated by confounding variables (such as age) and the coactivation of two additional endocrine systems (the adrenal androgenic system and the somatotropic growth axis) and requires further elucidation. In this paper, we present the design of, and preliminary observations from, the ongoing NIMH Intramural Longitudinal Study of the Endocrine and Neurobiological Events Accompanying Puberty. The aim of this study is to directly examine how the increase in sex steroid hormone production following activation of the HPG-axis (i.e., gonadarche) impacts neurodevelopment, and, additionally, to determine how gonadal development and maturation is associated with longitudinal changes in brain structure and function in boys and girls. To disentangle the effects of sex steroids from those of age and other endocrine events on brain development, our study design includes 1) selection criteria that establish a well-characterized baseline cohort of healthy 8-year-old children prior to the onset of puberty (e.g., prior to puberty-related sex steroid hormone production); 2) temporally dense longitudinal, repeated-measures sampling of typically developing children at 8-10 month intervals over a 10-year period between the ages of eight and 18; 3) contemporaneous collection of endocrine and other measures of gonadal, adrenal, and growth axis function at each timepoint; and 4) collection of multimodal neuroimaging measures at these same timepoints, including brain structure (gray and white matter volume, cortical thickness and area, white matter integrity, myelination) and function (reward processing, emotional processing, inhibition/impulsivity, working memory, resting-state network connectivity, regional cerebral blood flow). This report of our ongoing longitudinal study 1) provides a comprehensive review of the endocrine events of puberty; 2) details our overall study design; 3) presents our selection criteria for study entry (e.g., well-characterized prepubertal baseline) along with the endocrinological considerations and guiding principles that underlie these criteria; 4) describes our longitudinal outcome measures and how they specifically relate to investigating the effects of gonadal development on brain development; and 5) documents patterns of fMRI activation and resting-state networks from an early, representative subsample of our cohort of prepubertal 8-year-old children.

Neanderthal-Derived Genetic Variation is Associated with Functional Connectivity in the Brains of Living Humans.

Aim: To determine whether Neanderthal-derived genetic variation relates to functional connectivity patterns in the brains of living modern humans. Introduction: Nearly 50,000 years ago, Neanderthals interbred with ancestors of modern humans, imparting a genetic legacy that lives on today. The vestiges of this Neanderthal-derived genetic variation have been previously shown to be enriched in genes coding for neurogenesis and myelination and to alter skull shape and brain structure in living people. Materials and Methods: Using two independent cohorts totaling 553 healthy individuals, we employed multivariate distance matrix regression (MDMR) to determine whether any brain areas exhibited whole-brain functional connectivity patterns that significantly related to the degree of Neanderthal introgression. Identified clusters were then used as regions of interest in follow-up seed-based functional connectivity analyses to determine the connectivity patterns driving the relationships. Results: The MDMR analysis revealed that the percentage of Neanderthal-originating polymorphisms was significantly associated with the functional connectivity patterns of an area of the intraparietal sulcus (IPS) that was nearly identical in both cohorts. Using these IPS clusters as regions of interest in seed-based connectivity analyses, we found, again in both cohorts, that individuals with a higher proportion of Neanderthal-derived genetic variation showed increased IPS functional connectivity with visual processing regions, but decreased IPS connectivity with regions underlying social cognition. Conclusions: These findings demonstrate that the remnants of Neanderthal admixture continue to influence human brain function today, in ways that are consistent with anthropological conceptualizations of Neanderthal phenotypes, including the possibility that Neanderthals may have depended upon visual processing capabilities at the expense of social cognition, and this may have contributed to the extinction of this species through reduced cultural maintenance and inability to cope with fluctuating resources. This and other studies capitalizing on the emerging science surrounding ancient DNA provide a window through which to view an ancient lineage long past.

Longitudinal Positron Emission Tomography of Dopamine Synthesis in Subjects with GBA1 Mutations.

Mutations in GBA1, the gene mutated in Gaucher disease, are a common genetic risk factor for Parkinson disease, although the penetrance is low. We performed [18 F]-fluorodopa positron emission tomography studies of 57 homozygous and heterozygous GBA1 mutation carriers (15 with parkinsonism) and 98 controls looking for early indications of dopamine loss using voxelwise analyses to identify group differences in striatal [18 F]-fluorodopa uptake (Ki ). Forty-eight subjects were followed longitudinally. Cross-sectional and longitudinal comparisons of Ki and Ki change found significant effects of Parkinson disease. However, at baseline and over time, striatal [18 F]-fluorodopa uptake in mutation carriers without parkinsonism did not significantly differ from controls. ANN NEUROL 2020;87:652-657.

Distinct Polygenic Score Profiles in Schizophrenia Subgroups With Different Trajectories of Cognitive Development.

OBJECTIVE: Different cognitive development histories in schizophrenia may reflect variation across dimensions of genetic influence. The authors derived and characterized cognitive development trajectory subgroups within a schizophrenia sample and profiled the subgroups across polygenic scores (PGSs) for schizophrenia, cognition, educational attainment, and attention deficit hyperactivity disorder (ADHD). METHODS: Demographic, clinical, and genetic data were collected at the National Institute of Mental Health from 540 schizophrenia patients, 247 unaffected siblings, and 844 control subjects. Cognitive trajectory subgroups were derived through cluster analysis using estimates of premorbid and current IQ. PGSs were generated using standard methods. Associations were tested using general linear models and logistic regression. RESULTS: Cluster analyses identified three cognitive trajectory subgroups in the schizophrenia group: preadolescent cognitive impairment (19%), adolescent disruption of cognitive development (44%), and cognitively stable adolescent development (37%). Together, the four PGSs significantly predicted 7.9% of the variance in subgroup membership. Subgroup characteristics converged with genetic patterns. Cognitively stable individuals had the best adult clinical outcomes and differed from control subjects only in schizophrenia PGSs. Those with adolescent disruption of cognitive development showed the most severe symptoms after diagnosis and were cognitively impaired. This subgroup had the highest schizophrenia PGSs and had disadvantageous cognitive PGSs relative to control subjects and cognitively stable individuals. Individuals showing preadolescent impairment in cognitive and academic performance and poor adult outcome exhibited a generalized PGS disadvantage relative to control subjects and were the only subgroup to differ significantly in education and ADHD PGSs. CONCLUSIONS: Subgroups derived from patterns of premorbid and current IQ showed different premorbid and clinical characteristics, which converged with broad genetic profiles. Simultaneous analysis of multiple PGSs may contribute to useful clinical stratification in schizophrenia.