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Auditory processing is widely understood to occur differently in autism, though the patterns of brain activity underlying these differences are not well understood. The diversity of autism also means brain-wide networks may change in various ways to produce similar behavioral outputs. We used larval zebrafish to investigate auditory habituation in four genetic lines relevant to autism: fmr1 , mecp2 , scn1lab and cntnap2 . In free-swimming behavioral tests, we found each line had a unique profile of auditory hypersensitivity and/or delayed habituation. Combining the optical transparency of larval zebrafish with genetically encoded calcium indicators and light-sheet microscopy, we then observed brain-wide activity at cellular resolution during auditory habituation. As with behavior, each line showed unique alterations in brain-wide spontaneous activity, auditory processing, and adaptation in response to repetitive acoustic stimuli. We also observed commonalities in activity across our genetic lines that indicate shared circuit changes underlying certain aspects of their behavioral phenotypes. These were predominantly in regions involved in sensory integration and sensorimotor gating rather than primary auditory areas. Overlapping phenotypes include differences in the activity and functional connectivity of the telencephalon, thalamus, dopaminergic regions, and the locus coeruleus, and excitatory/inhibitory imbalance in the cerebellum. Unique phenotypes include loss of activity in the habenula in scn1lab , increased activity in auditory regions in fmr1, and differences in network activity over time in mecp2 and cntnap2 . Comparing these distinct but overlapping brain-wide auditory networks furthers our understanding of how diverse genetic factors can produce similar behavioral effects through a range of circuit- and network-scale mechanisms.

Enlargement of the cerebrospinal fluid (CSF)-filled brain ventricles (cerebral ventriculomegaly), the cardinal feature of congenital hydrocephalus (CH), is increasingly recognized among patients with autism spectrum disorders (ASD). KATNAL2, a member of Katanin family microtubule-severing ATPases, is a known ASD risk gene, but its roles in human brain development remain unclear. Here, we show that nonsense truncation of Katnal2 ( Katnal2Δ 17 ) in mice results in classic ciliopathy phenotypes, including impaired spermatogenesis and cerebral ventriculomegaly. In both humans and mice, KATNAL2 is highly expressed in ciliated radial glia of the fetal ventricular-subventricular zone as well as in their postnatal ependymal and neuronal progeny. The ventriculomegaly observed in Katnal2 Δ17 mice is associated with disrupted primary cilia and ependymal planar cell polarity that results in impaired cilia-generated CSF flow. Further, prefrontal pyramidal neurons in ventriculomegalic Katnal2 Δ 17 mice exhibit decreased excitatory drive and reduced high-frequency firing. Consistent with these findings in mice, we identified rare, damaging heterozygous germline variants in KATNAL2 in five unrelated patients with neurosurgically treated CH and comorbid ASD or other neurodevelopmental disorders. Mice engineered with the orthologous ASD-associated KATNAL2 F244L missense variant recapitulated the ventriculomegaly found in human patients. Together, these data suggest KATNAL2 pathogenic variants alter intraventricular CSF homeostasis and parenchymal neuronal connectivity by disrupting microtubule dynamics in fetal radial glia and their postnatal ependymal and neuronal descendants. The results identify a molecular mechanism underlying the development of ventriculomegaly in a genetic subset of patients with ASD and may explain persistence of neurodevelopmental phenotypes in some patients with CH despite neurosurgical CSF shunting., Competing Interests: Competing interests statement:The authors declare no competing interest.

Here, we present Brain Registration and Evaluation for Zebrafish (BREEZE)-mapping, a user-friendly pipeline for the registration and analysis of whole-brain images in larval zebrafish. We describe steps for pre-processing, registration, quantification, and visualization of whole-brain phenotypes in zebrafish mutants of genes associated with neurodevelopmental and neuropsychiatric disorders. By utilizing BioImage Suite Web, an open-source software package originally developed for processing human brain imaging data, we provide a highly accessible whole-brain mapping protocol developed for users with general computational proficiency. For complete details on the use and execution of this protocol, please refer to Weinschutz Mendes et al. (2023). 1 ., Competing Interests: Declaration of interests X.P. is a consultant for the Brain Electrophysiology Laboratory Company. X.P. also consults and has an ownership stake in Veridat., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Motor stereotypies are common in children with autism spectrum disorder (ASD), intellectual disability, or sensory deprivation, as well as in typically developing children ("primary" stereotypies, pCMS). The precise pathophysiological mechanism for motor stereotypies is unknown, although genetic etiologies have been suggested. In this study, we perform whole-exome DNA sequencing in 129 parent-child trios with pCMS and 853 control trios (118 cases and 750 controls after quality control). We report an increased rate of de novo predicted-damaging DNA coding variants in pCMS versus controls, identifying KDM5B as a high-confidence risk gene and estimating 184 genes conferring risk. Genes harboring de novo damaging variants in pCMS probands show significant overlap with those in Tourette syndrome, ASD, and those in ASD probands with high versus low stereotypy scores. An exploratory analysis of these pCMS gene expression patterns finds clustering within the cortex and striatum during early mid-fetal development. Exploratory gene ontology and network analyses highlight functional convergence in calcium ion transport, demethylation, cell signaling, cell cycle and development. Continued sequencing of pCMS trios will identify additional risk genes and provide greater insights into biological mechanisms of stereotypies across diagnostic boundaries., Competing Interests: I have read the journal’s policy, and the authors of this manuscript have the following competing interests: Dr. Fernandez receives research/grant support from the National Institutes of Mental Health. Dr. Olfson receives research support from the National Institutes of Mental Health, the Alan B. Slifka Foundation through the Riva Ariella Ritvo endowment, and the International Obsessive-Compulsive Disorder Foundation. Dr. Singer serves as a consultant for Abide Therapeutics, Inc; Cello Health BioConsulting; ClearView Healthcare Partners; Teva Pharmaceutical Industries Ltd; and Trinity Partners, LLC. Dr. Singer receives publishing royalties from Elsevier and research/grant support from the Tourette Association of America. Other authors declare no potential conflicts. This does not alter our adherence to PLOS ONE policies on sharing data and materials., (Copyright: © 2023 Fernandez et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Advancing from gene discovery in autism spectrum disorders (ASDs) to the identification of biologically relevant mechanisms remains a central challenge. Here, we perform parallel in vivo functional analysis of 10 ASD genes at the behavioral, structural, and circuit levels in zebrafish mutants, revealing both unique and overlapping effects of gene loss of function. Whole-brain mapping identifies the forebrain and cerebellum as the most significant contributors to brain size differences, while regions involved in sensory-motor control, particularly dopaminergic regions, are associated with altered baseline brain activity. Finally, we show a global increase in microglia resulting from ASD gene loss of function in select mutants, implicating neuroimmune dysfunction as a key pathway relevant to ASD biology., Competing Interests: Declaration of interests X.P. is a consultant for the Brain Electrophysiology Laboratory Company., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Autism spectrum disorders (ASDs) are a group of neurodevelopmental disorders associated with deficits in social communication and restrictive, repetitive patterns of behavior, that affect up to 1 in 54 children. ASDs clearly demonstrate a male bias, occurring ~4 times more frequently in males than females, though the basis for this male predominance is not well-understood. In recent years, ASD risk gene discovery has accelerated, with many whole-exome sequencing studies identifying genes that converge on common pathways, such as neuronal communication and regulation of gene expression. ASD genetics studies have suggested that there may be a "female protective effect," such that females may have a higher threshold for ASD risk, yet its etiology is not well-understood. Here, we review common biological pathways implicated by ASD genetics studies as well as recent analyses of sex differential processes in ASD using imaging genomics, transcriptomics, and animal models. Additionally, we discuss recent investigations of ASD risk genes that have suggested a potential role for estrogens as modulators of biological pathways in ASD, and highlight relevant molecular and cellular pathways downstream of estrogen signaling as potential avenues for further investigation., Competing Interests: 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., (Copyright © 2021 Enriquez, Gupta and Hoffman.)

Hundreds of human genes are associated with neurological diseases, but translation into tractable biological mechanisms is lagging. Larval zebrafish are an attractive model to investigate genetic contributions to neurological diseases. However, current CRISPR-Cas9 methods are difficult to apply to large genetic screens studying behavioural phenotypes. To facilitate rapid genetic screening, we developed a simple sequencing-free tool to validate gRNAs and a highly effective CRISPR-Cas9 method capable of converting >90% of injected embryos directly into F0 biallelic knockouts. We demonstrate that F0 knockouts reliably recapitulate complex mutant phenotypes, such as altered molecular rhythms of the circadian clock, escape responses to irritants, and multi-parameter day-night locomotor behaviours. The technique is sufficiently robust to knockout multiple genes in the same animal, for example to create the transparent triple knockout crystal fish for imaging. Our F0 knockout method cuts the experimental time from gene to behavioural phenotype in zebrafish from months to one week., Competing Interests: FK, GP, MG, GG, PA, TH, HT, SL, HD, JF, DW, ED, SW, EH, JR No competing interests declared, (© 2021, Kroll et al.)

Congenital hydrocephalus (CH), characterized by enlarged brain ventricles, is considered a disease of excessive cerebrospinal fluid (CSF) accumulation and thereby treated with neurosurgical CSF diversion with high morbidity and failure rates. The poor neurodevelopmental outcomes and persistence of ventriculomegaly in some post-surgical patients highlight our limited knowledge of disease mechanisms. Through whole-exome sequencing of 381 patients (232 trios) with sporadic, neurosurgically treated CH, we found that damaging de novo mutations account for >17% of cases, with five different genes exhibiting a significant de novo mutation burden. In all, rare, damaging mutations with large effect contributed to ~22% of sporadic CH cases. Multiple CH genes are key regulators of neural stem cell biology and converge in human transcriptional networks and cell types pertinent for fetal neuro-gliogenesis. These data implicate genetic disruption of early brain development, not impaired CSF dynamics, as the primary pathomechanism of a significant number of patients with sporadic CH.

Zebrafish are increasingly being utilized as a model system to investigate the function of the growing list of risk genes associated with neurodevelopmental disorders. This is due in large part to the unique features of zebrafish that make them an optimal system for this purpose, including rapid, external development of transparent embryos, which enable the direct visualization of the developing nervous system during early stages, large progenies, which provide considerable tractability for performing high-throughput pharmacological screens to identify small molecule suppressors of simple behavioral phenotypes, and ease of genetic manipulation, which has been greatly facilitated by the advent of CRISPR/Cas9 gene editing technologies. This review article focuses on studies that have harnessed these advantages of the zebrafish system for the functional analysis of genes that are strongly associated with the following neurodevelopmental disorders: autism spectrum disorders (ASD), epilepsy, intellectual disability (ID) and schizophrenia. We focus primarily on studies describing early morphological and behavioral phenotypes during embryonic and larval stages resulting from loss of risk gene function. We highlight insights into basic mechanisms of risk gene function gained from these studies as well as limitations of studies to date. Finally, we discuss advances in in vivo neural circuit imaging in zebrafish, which promise to transform research using the zebrafish model by illuminating novel circuit-level mechanisms with relevance to neurodevelopmental disorders.

Autism spectrum disorders (ASDs) are a group of devastating neurodevelopmental syndromes that affect up to 1 in 68 children. Despite advances in the identification of ASD risk genes, the mechanisms underlying ASDs remain unknown. Homozygous loss-of-function mutations in Contactin Associated Protein-like 2 (CNTNAP2) are strongly linked to ASDs. Here we investigate the function of Cntnap2 and undertake pharmacological screens to identify phenotypic suppressors. We find that zebrafish cntnap2 mutants display GABAergic deficits, particularly in the forebrain, and sensitivity to drug-induced seizures. High-throughput behavioral profiling identifies nighttime hyperactivity in cntnap2 mutants, while pharmacological testing reveals dysregulation of GABAergic and glutamatergic systems. Finally, we find that estrogen receptor agonists elicit a behavioral fingerprint anti-correlative to that of cntnap2 mutants and show that the phytoestrogen biochanin A specifically reverses the mutant behavioral phenotype. These results identify estrogenic compounds as phenotypic suppressors and illuminate novel pharmacological pathways with relevance to autism., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Autism spectrum disorder (ASD) is a complex developmental syndrome of unknown etiology. Recent studies employing exome- and genome-wide sequencing have identified nine high-confidence ASD (hcASD) genes. Working from the hypothesis that ASD-associated mutations in these biologically pleiotropic genes will disrupt intersecting developmental processes to contribute to a common phenotype, we have attempted to identify time periods, brain regions, and cell types in which these genes converge. We have constructed coexpression networks based on the hcASD "seed" genes, leveraging a rich expression data set encompassing multiple human brain regions across human development and into adulthood. By assessing enrichment of an independent set of probable ASD (pASD) genes, derived from the same sequencing studies, we demonstrate a key point of convergence in midfetal layer 5/6 cortical projection neurons. This approach informs when, where, and in what cell types mutations in these specific genes may be productively studied to clarify ASD pathophysiology., (Copyright © 2013 Elsevier Inc. All rights reserved.)

In this paper, we demonstrate the application of Metal-Assisted and Microwave-Accelerated Evaporative Crystallization (MA-MAEC) technique to rapid and selective crystallization of a small drug compound. i.e. acetaminophen. Subsequent characterization of the crystals by optical microscopy, powder X-ray diffraction (PXRD) and Raman spectroscopy showed quantitatively selective growth of different crystal forms at various experimental conditions. Acetaminophen crystals were grown predominantly as Form I (99%) on blank glass slides at room temperature. Form II crystals with 39% purity grown on SIFs using microwave energy.

Background: It has previously been shown that specific microdeletions and microduplications, many of which also associated with cognitive impairment (CI), can present with autism spectrum disorders (ASDs). Multiplex ligation-dependent probe amplification (MLPA) represents an efficient method to screen for such recurrent microdeletions and microduplications., Methods: In the current study, a total of 279 unrelated subjects ascertained for ASDs were screened for genomic disorders associated with CI using MLPA. Fluorescence in situ hybridization (FISH), quantitative polymerase chain reaction (Q-PCR) and/or direct DNA sequencing were used to validate potential microdeletions and microduplications. Methylation-sensitive MLPA was used to characterize individuals with duplications in the Prader-Willi/Angelman (PWA) region., Results: MLPA showed two subjects with typical ASD-associated interstitial duplications of the 15q11-q13 PWA region of maternal origin. Two additional subjects showed smaller, de novo duplications of the PWA region that had not been previously characterized. Genes in these two novel duplications include GABRB3 and ATP10A in one case, and MKRN3, MAGEL2 and NDN in the other. In addition, two subjects showed duplications of the 22q11/DiGeorge syndrome region. One individual was found to carry a 12 kb deletion in one copy of the ASPA gene on 17p13, which when mutated in both alleles leads to Canavan disease. Two subjects showed partial duplication of the TM4SF2 gene on Xp11.4, previously implicated in X-linked non-specific mental retardation, but in our subsequent analyses such variants were also found in controls. A partial duplication in the ASMT gene, located in the pseudoautosomal region 1 (PAR1) of the sex chromosomes and previously suggested to be involved in ASD susceptibility, was observed in 6-7% of the cases but in only 2% of controls (P = 0.003)., Conclusion: MLPA proves to be an efficient method to screen for chromosomal abnormalities. We identified duplications in 15q11-q13 and in 22q11, including new de novo small duplications, as likely contributing to ASD in the current sample by increasing liability and/or exacerbating symptoms. Our data indicate that duplications in TM4SF2 are not associated with the phenotype given their presence in controls. The results in PAR1/PAR2 are the first large-scale studies of gene dosage in these regions, and the findings at the ASMT locus indicate that further studies of the duplication of the ASMT gene are needed in order to gain insight into its potential involvement in ASD. Our studies also identify some limitations of MLPA, where single base changes in probe binding sequences alter results. In summary, our studies indicate that MLPA, with a focus on accepted medical genetic conditions, may be an inexpensive method for detection of microdeletions and microduplications in ASD patients for purposes of genetic counselling if MLPA-identified deletions are validated by additional methods.

Unlabelled: Image quality in PET is typically assessed using measures such as contrast recovery, noise variation, and signal-to-noise ratio (SNR). However, these criteria do not directly reflect performance in the clinical use of the images. Lesion detection is a critical task in the clinical interpretation of many PET studies. A receiver operating characteristic (ROC) study is an accepted method for quantitatively evaluating detection performance with respect to factors that influence image quality. ROC and localization ROC (LROC) analyses were conducted to investigate the effects of lesion contrast, SNR, and size on detectability of hot lesions in PET images., Methods: A thorax phantom was imaged with spheres of 3 sizes simulating lesions (0.45, 1.0, and 1.9 mL). The relative activity in the lesions and the total number of counts acquired were each varied by factors of 2 to ascertain the effects of contrast and SNR, respectively. Measured attenuation correction and a standard reconstruction protocol were used. Three nuclear medicine physicians and 6 medical physicists participated as readers, rating each image and indicating the suspected lesion location. The area under the calculated ROC and LROC curves (Az and Az,LROC) were used as measures of detection performance., Results: Detection performance was shown to increase from virtually random (Az approximately 0.5, Az,LROC approximately 0.2) to superior (Az > 0.9, Az,LROC > 0.9) as lesion contrast was increased by 50% and as lesion SNR was doubled. Detection performance was not seen to vary when comparison was made using image-based measures alone., Conclusion: This study quantitatively shows that moderate increases in the image-based measures of lesion contrast and SNR give a relatively large increase in the task-based measure of lesion detection as measured by ROC and LROC analyses. Thus, techniques that give modest increases in lesion contrast or SNR are expected to improve detection. Results will be useful in evaluating improvement in detection for various reconstruction, acquisition, and data analysis methods that enhance contrast or noise performance.

Unlabelled: Receiver operating characteristic (ROC) and localization ROC (LROC) studies were performed to compare lesion detection at the borderline of detectability on images reconstructed with two-dimensional filtered backprojection (FBP) without attenuation correction (a common clinical protocol), three-dimensional FBP without attenuation correction, two-dimensional FBP with segmented attenuation correction and a two-dimensional iterative maximum a posteriori (MAP) algorithm using attenuation correction. Lung cancer was the model for the study because of the prominent role of 18F-fluorodeoxyglucose PET in the staging of lung cancer and the importance of lesion detection for staging., Methods: Simulated lung cancer lesions were added to two-dimensional and three-dimensional PET data from healthy volunteers. Data were reconstructed using the four methods. Four nuclear medicine physicians evaluated the images. Detection performance with each method was compared using ROC and LROC analysis. Jackknife analysis provided estimates of statistical significance for differences across all readers for the ROC results., Results: ROC and LROC results indicated statistically significant degradation in detection performance with three-dimensional acquisition (average area under ROC curves [Az] 0.51; average area under LROC curves [A(z,LROC)] 0.13) and segmented attenuation correction (average Az 0.59; average Az,LROC 0.29) compared with two-dimensional FBP without attenuation correction (average Az 0.79; average A(z,LROC) 0.54). ROC and LROC results indicated an improvement in detection performance with iterative MAP reconstruction (average Az 0.83; average A(z,LROC) 0.64) compared with two-dimensional FBP reconstruction; this improvement was not statistically significant., Conclusion: Use of segmented attenuation correction or three-dimensional acquisition with FBP reconstruction is not expected to improve detection of lung lesions on whole-body PET images compared with images with two-dimensional FBP without attenuation correction. The potential improvement in detection obtained with an iterative MAP reconstruction method is small compared with that obtained with two-dimensional FBP without attenuation correction.

The sensitivity and resolution of a new 3D modular BGO detector system for positron emission tomography (PET) was examined in terms of its effect on the signal to noise ratio in the final image. The detector system uses custom hardware and firmware to adjust sensitivity and resolution to achieve optimum performance. This system utilizes lookup up tables (LUT) containing individually tailored positioning and energy windows to assure that events are correctly distributed among the detector elements. Without customizing the LUT, and for a common energy window, sensitivity was found to vary as much as a factor of 3.5 and resolution as much as 50% from center to edge within a single detector module. Eight different LUT configurations were tested, varying both energy window and positioning criteria. Typical intrinsic resolutions and sensitivities of the detector elements were determined in all configurations. The effect of nonuniformity of sensitivity of the detector system on image noise was evaluated by imaging uniform cylinders over a wide range of accumulated events. The effect of counting statistics in the measurement of detector sensitivities for the normalization of sinogram data was determined independently. The best of the LUT configurations provided an 16% improvement in effective sensitivity, while also providing a 10% improvement in the average intrinsic spatial resolution relative to the default configuration of the PET system.

The results of a receiver operator characteristic (ROC) study comparing maximum likelihood estimator (MLE) reconstructions of human FDG PET brain scan data to filtered backprojection reconstructions of the same data are reported. The purpose of the study was to determine whether MLE reconstructions would result in higher detectability of small focal lesions introduced artificially into otherwise normal scan data. One physician assisted in defining the location and intensity of the lesions and five physicians read the final images. Data from 90 datasets were used for the study. Of those, 42 were left in their original "normal" condition and 48 were modified by added lesions. All datasets were reconstructed by the two methods and submitted to the five physicians for evaluation. The results show an increase in the area under the ROC curve from approximately 0.65 for filtered backprojection to approximately 0.71 for the maximum likelihood reconstructions for four of the five observers with good statistical significance.

Large sensitivity increases are realized in positron emission tomography when the interplane septa are removed and all lines of response acquired. Unfortunately, three-dimensional acquisition results in a large increase in scatter fraction which prevents accurate quantitation. By acquiring short two-dimensional scans prior to three-dimensional ones, scatter distributions can be estimated from differences between lines of response common to both datasets. This initial scatter distribution can be further modified to approximate scatter in the entire three-dimensional dataset. The method was validated with phantom measurements in which absolute activity concentrations were known in all compartments. Following scatter correction, a four-compartment phantom that was nonuniform in activity and density, both axially and transaxially, gave activity concentrations of 0.45 +/- 0.02, 0.31 +/- 0.02, 0.01 +/- 0.01 and 0.01 +/- 0.01 microCi/cc for compartments containing 0.43, 0.29, 0.0 (air) and 0.0 (water) microCi/cc, respectively. Thus, scatter distributions for complex sources can be estimated from image data without lengthy Monte-Carlo simulations. When activity distributions vary slowly with time, this method can be used to correct for scatter in three-dimensional patient studies.

The work presented evaluates the statistical characteristics of regional bias and expected error in reconstructions of real positron emission tomography (PET) data of human brain fluoro-deoxiglucose (FDG) studies carried out by the maximum likelihood estimator (MLE) method with a robust stopping rule, and compares them with the results of filtered backprojection (FBP) reconstructions and with the method of sieves. The task of evaluating radioisotope uptake in regions-of-interest (ROIs) is investigated. An assessment of bias and variance in uptake measurements is carried out with simulated data. Then, by using three different transition matrices with different degrees of accuracy and a components of variance model for statistical analysis, it is shown that the characteristics obtained from real human FDG brain data are consistent with the results of the simulation studies.

Methods for whole-body PET imaging have been developed to provide a clinical tool for the detection and evaluation of primary and metastatic cancers. The axial FOV of the PET system is extended by imaging at multiple bed positions to cover the whole body. In typical rectilinear PET scans, only a small fraction of the data is collected to form two-dimensional projection images. In this work, 100% of the projection data was collected to form the two-dimensional projection images. These projection images were generated for continuous angles over 180 degrees by resorting sinogram data. In addition, tomographic images were formed by using filtered backprojection reconstruction without attenuation correction. Coronal and sagittal cuts were then extracted from the three-dimensional data set. The tomographic images were reconstructed to a resolution of 10.8 mm in all dimensions because of statistical limitations of the data. Both methods of image formation resulted in images of high quality with the tomographic reconstruction providing the highest contrast and resolution. An acquisition time of 1-2 min/bed position after a 10-mCi injection of [18F]fluoride ion or [18F]FDG was found to give a sufficient number of counts for producing images of good resolution and contrast, from a total scanning time of 32-64 min.

The design features of a PET system designed for animal studies are described and its performance evaluated. The system employs a two-dimensional modular detector array consisting of bismuth germanate detector elements that are 3.5 mm (transaxially) by 6.25 mm (axially) by 30 mm (deep). These arrays are optically coupled to a pair of dual-photo-multiplier tubes (PMT). The detector ring is 64 cm in diameter with a field of view (FOV) of 40 cm by 5.4 cm axially, acquiring 15 slices at 3.4 mm spacing. These features include: (1) digitization of PMT signals from each block for improved position and energy discrimination of coincident events and (2) dual-window energy discrimination for simultaneous but separate acquisition of photopeak and scatter data. Intrinsic resolution averages 3.5 mm at the center of the FOV, while reconstructed resolution (ramp filter) ranges from 3.8 mm at the center of the FOV to 4.6 mm at an 8 cm radius. Axial resolution averages 4.4 and 4.9 mm and sensitivity averages 4.2 and 6.1 kcps/microCi/cc for cross planes and enhanced direct planes, respectively. Randoms fraction is high due to reduced interplane shielding, giving a peak true count rate of 103 kcps for a 10 cm cylinder. Scatter as a fraction of trues is 16% for a 10 cm cylinder at a lower energy threshold of 350 keV. All parameters are sensitive to energy threshold. Spatial resolution improves by 11% transaxially and 9% axially, scatter fraction drops to 10%, and overall sensitivity drops by 48% when the threshold value is increased from 350 keV to 450 keV.

A method using the activity in the abdominal aorta of human and animal subjects to noninvasively estimate blood-pool input function in dynamic, abdominal PET scans is proposed and validated in this paper. Partial volume effects due to the aorta's dimensions are corrected by a semi-automated algorithm based on the transaxial resolution in the reconstructed images. The technique was validated by comparing PET measurements of abdominal aortic activity to well counter measurements of arterial blood samples (eight canine renal studies) and to PET measurements of left ventricular cavity activity (eight human hepatic studies). In renal studies, correlation analysis of the areas subtended by the two input functions yielded an essentially unitary slope (1.03 +/- 0.09), with high correlation (R2 greater than 0.95, p less than 0.001). In hepatic studies, similar values (0.99 +/- 0.03 and R2 greater than 0.85, p less than 0.001) were found. Correlation of the blood flow estimates based on the two input functions and a two-compartment model produced slopes of 1.07 +/- 0.16 and 1.03 +/- 0.07, and correlations of (R2 greater than 0.98, p less than 0.001) and (R2 greater than 0.97, p less than 0.001) for the renal and hepatic studies, respectively. We conclude that noninvasive, accurate measurements of the arterial input function by dynamic PET imaging are possible and represent a clinically viable alternative to arterial blood sampling.

The quantitative imaging characteristics of ultrahigh-resolution parallel-hole SPECT, including 3-D geometric detector response, attenuation, scatter, and statistical noise, were investigated by simulations based on a complex digitized 3-D brain model of the gray and white matter distributions. The projection data resulting from a uniform distribution of gray and white matter radioactivity, in a ratio of 5:1, were simulated. The results demonstrate significant qualitative and quantitative artifacts in reconstructed human brain images. In the absence of attenuation, scatter, and noise, artifactual variation caused inaccuracies in regional radioactivity quantification. Inclusion of attenuation scatter, and noise in the simulation caused additional artifacts, and resulted in reconstructed images which qualitatively and quantitatively corresponded very closely to reconstructed images of the actual 3-D brain phantom which was constructed from the same set of data as the mathematical 3-D brain model. It is concluded that the major degrading factor in SPECT neuroimaging is the 3-D geometric detector response function.

A standard set of performance measurements is proposed for use with positron emission tomographs. This set of measurements has been developed jointly by the Computer and Instrumentation Council of the Society of Nuclear Medicine and the National Electrical Manufacturers Association. The measurements include tests of spatial resolution, scatter fraction, sensitivity, count rate losses and randoms, uniformity, scatter correction, attenuation correction, and count rate linearity correction.

We have developed a three-dimensional computer simulation of SPECT imaging. We have applied the simulation procedure to the realistic mathematical Hoffman three-dimensional brain model to generate the projection data (in the absence of attenuation, scatter, or noise) of both a parallel-hole and a multidetector SPECT system with point-focusing collimators. The simulated projection data were then reconstructed using standard software. The projection data resulting from the distribution of grey matter alone, or grey and white matter, were simulated. The results of these simulations indicate the existence of significant qualitative and quantitative artifacts in reconstructed human brain images. For example, the reconstructed values for grey matter along a cortical circumferential profile in a transverse slice through the basal ganglia varied by a factor of 2.40 (parallel-hole) and 2.99 (point-focusing), although the original grey matter values were identical in all cortical regions in the model. We have compared the simulated reconstructed images with those obtained by imaging the physical three-dimensional Hoffman brain phantom, which was constructed based upon the same set of data from which the mathematical three-dimensional Hoffman brain model was derived. Although the simulation did not include all of the degrading factors present in the physical imaging, the two images were in good agreement, indicating the applicability of the simulation to a realistic situation and the importance of the detector resolution effect.

Two methods of rejecting pileup events in 2-D array detectors used in positron emission tomography (PET) are investigated. Since the methods are based on different physical properties, they were investigated separately and in combination. Method one is based on the fact that valid events in the 2-D array detector occur only in a limited pulseheight range for each detector element, and by rejecting events outside these energy ranges, many of the pileup events can be rejected. Method two is based on the fact that most pileup event pairs have a time differential, which will cause a difference in the shape of the signal which increases the rise time of an integrated signal or the crossover time of a bipolar signal from a shaping amplifier. Method two proved to be somewhat better than method one. However, the ideal system would most likely use both methods simultaneously.

A fully 3-D reconstruction algorithm has been developed to reconstruct data from a 16 ring PET camera (a Siemens/CTI 953B) with automatically retractable septa. The tomograph is able to acquire coincidences between any pair of detector rings and septa retraction increases the total system count rate by a factor of 7.8 (including scatter) and 4.7 (scatter subtracted) for a uniform, 20 cm diameter cylinder. The reconstruction algorithm is based on 3-D filtered backprojection, expressed in a form suitable for the multi-angle sinogram data. Sinograms which are not measured due to the truncated cylindrical geometry of the tomograph, but which are required for a spatially invariant response function, are obtained by forward projection. After filtering, the complete set of sinograms is backprojected into a 3-D volume of 128x128x31 voxels using a voxel-driven procedure. The algorithm has been validated with simulation, and tested with both phantom and clinical data from the 953B.

13N-labeled ammonia was used to investigate 1) the cerebral extraction and clearance of ammonia, 2) the mechanism by which capillaries accommodate changes in cerebral blood flow (CBF) and 3) its use for the measurement of CBF. The unidirectional extraction of 13NH3 in rhesus monkeys was measured during PaCO2 induced changes in CBF and dog studies were performed using in vitro tissue counting techniques to examine 13NH3 extraction in gray and white matter, mixed tissue and cerebellum during variations in CBF produced by combinations of embolization, local brain compression, and changes in PaCO2. The single pass extraction fraction of 13NH3 varied from about 70 to 20% over a CBF range of 12 to 140 cc/min/100 g. Capillary permeability-surface area product (PS) estimates with a Renkin/Crone model show PS increasing with CBF. The magnitude and rate of increase in PS with CBF was highest in gray matter greater than mixed tissue greater than white matter. Tissue extraction of 13NH3 vs CBF relationship was best described by a unidirectional transport model in which CBF increases by both recruitment of capillaries and by increases of blood velocity in open capillaries. This saturable-recruitment model provides a possible explanation for the mechanism of flow changes at the capillary level. The net 13NH3 extraction subsequent to an i.v. injection increases non-linearly with CBF. Doubling or halving basal CBF produced from 35 to 50% changes in the 13N tissue concentrations with further increases in CBF associated with progressively smaller changes in 13N concentrations.

To evaluate accurately the imaging characteristics of positron emission tomography (PET), a realistic computer-simulated brain phantom was developed. A cross-sectional slice from a human cadaver brain was chosen for its combination of gray matter, white matter, and cerebrospinal fluid (CSF) regions. The slice was photographed and digitized into a gray-level image with a video digitizer, boundary edges were located around cerebral structures in the digitized image, and each structural region was assigned a uniform pixel value dependent on both the cerebral parameter (e.g., blood flow, oxygen uptake, metabolic rate) under investigation and the type of structure (gray matter, white matter, CSF). Line integrals through the regions were generated at various angular and transverse positions according to specific physical characteristics (such as detector line-spread function) of a tomographic scanner configuration to create a set of simulated but realistic projection measurements. The set of projection measurements can be processed with any standard reconstruction program to create a tomographic image to reveal the effects of various PET characteristics. Investigations with this computer-simulated brain phantom have demonstrated its usefulness for examining the interrelations among neuroanatomical structure volume, tomographic spatial resolution, partial volume effect, and nonlinear parameter estimation. Transportability of the simulated phantom and the procedure to other medical imaging environments is described, and limitations of this simulation procedure are discussed.

Positron emission tomography (PET) was applied to the measurement of myocardial perfusion using the perfusion tracer 13N-labeled ammonia. 13N ammonia was delivered intravenously to 13 healthy volunteers both at rest and during supine bicycle exercise. Dynamic PET imaging was obtained in three cross-sectional planes for 10 minutes commencing with each injection. The left ventricle was divided into eight sectors, and a small region of interest was assigned to the left ventricular blood pool to obtain the arterial input function. The net extraction of 13N ammonia was obtained for each sector by dividing the tissue 13N concentration at 10 minutes by the integral of the input function from the time of injection to 10 minutes. With this approach for calculating net extractions, rest and exercise net extractions were not significantly different from each other. To obviate possible overestimation of the true 13N ammonia input function by contamination by 13N-labeled compounds other than 13N ammonia or by spillover from myocardium into blood pool, the net extractions were calculated using only the first 90 seconds of the blood and tissue time-activity curves. This approach for calculating net extractions yielded significant differences between rest and exercise, with an average ratio of exercise to rest of 1.38 +/- 0.34. Nonetheless, the increase was less than predicted from the average 2.7-2.8-fold increase in double product at peak exercise or the 1.7-fold increase in double product at 1 minute after exercise. However, when the first 90 seconds of dynamic data were fit with a two compartment tracer kinetic model, average perfusion rates of 0.75 +/- 0.43 ml/min/g at rest and 1.50 +/- 0.74 ml/min/g with exercise were obtained. This average increase in perfusion of 2.2-fold corresponded to similar average increases in double product. Thus, the noninvasive technique of PET imaging with 13N ammonia shows promise for future applications in determining absolute flows in patients with coronary artery disease.

We describe a double-tracer technique for positron computed tomography (PCT) that utilizes the differences in physical decay rates to distinguish between two positron emitters. The technique is applied to PCT studies on phantoms (a) to separate two tracers with different half-lives, and (b) to measure simultaneously the attenuation coefficients and the tracer concentrations in a phantom. Results from these experiments are encouraging, but they also reveal a major limitation of the technique, namely, noise enhancement that is related to the relative half-lives of the two tracers and the time duration of the PCT scans, as well as to the relative concentrations of the two tracers. If these parameters are carefully selected, the double-tracer technique will have high potential for examining simultaneously two different biological processes with PCT and for measuring tracer concentrations and attenuation correction factors at the same time.

To examine the use of 2-deoxy-2-[18F]fluoro-D-glucose (2-FDG) as a glucose analog for measuring glucose utilization rate in myocardium, dual-tracer kinetic experiments with 2-FDG and 2-[3H]glucose were performed in the perfused, isolated rabbit interventricular septum to measure simultaneously the transport and phosphorylation rates of glucose and 2-FDG. Results of the present study indicated that, in the septum, the transport rate constants of 2-FDG and glucose were similar in magnitude, the phosphorylation rate constant for 2-FDG was about 60% of that of glucose, hypoxia caused an increase in phosphorylation rates of glucose and 2-FDG without affecting transport.

To validate the determination of the arterial input function by noninvasive dynamic PET imaging, measurements of blood-pool activity in canine LV by PET were compared to beta probe measurements of arterial blood withdrawn directly from the LV. PET scans were done during intravenous bolus injections of [13N]ammonia or 82Rb, while the activity of blood withdrawn continuously from a catheter inserted in the LV was measured with a beta probe. PET determinations of LV blood-pool activity were compared with dispersion-corrected beta probe time-activity curves. In 15 experiments involving four dogs under a wide range of physiologic conditions, LV time-activity curves obtained with PET matched well in shape with those obtained with the beta probe. Linear regression yielded slopes within 10% of unity (95% confidence interval) and high correlation (r greater than 0.968, p less than 0.001). We conclude that noninvasive measurement of the arterial input function by dynamic PET imaging is valid.

Radiopharmaceuticals incorporated directly into the metabolic pathways in myocardium provide a useful means for evaluating such processes. Palmitic acid, a major physiologic substrate of myocardium, has a well-understood role in myocardial metabolism. Accordingly, 11C-palmitic acid was the substrate chosen for use in conjunction with positron emission transaxial tomography to obtain images of canine myocardium. This procedure provides high-contrast images of tranverse sections of the myocardium, with good target-to-nontarget ratios (in the image), over a period of 5 to 85 minutes. Clearance half-times for blood and myocardial tissue were found to be 4.8 and 330 min, respectively. In normal myocardium, images obtained with 11C-palmitic acid were those obtained with 13NH3 and 11CO-hemoglobin. In vivo images of hearts with myocardial infarcts showed a clear delineration of infarcts and normal tissue.

To characterize externally detectable changes in myocardial metabolism of free fatty acids (FFA) and glucose associated with ischemia, isovolumically beating rabbit hearts were perfused under conditions of selected flows with cyclotron-produced, short-lived (t1/2 - 20.4 minutes), 11C-labeled isotopes of glucose and FFA. Tension-time index decreased 83% and lactate production increased from 0.5 +/- 1.9 (SE) to 5.3 +/- 2.1 mumol/min per g of dry weight reflecting myocardial ischemia after flow was reduced from 20 to 5 ml/min. After 30 minutes of low flow the myocardial accumulation of 11C-octanoate, expressed as the extraction fraction, declined from 56 +/- 15% to 30 +/- 3%, reflecting metabolic suppression of FFA extraction during low flow. Effects attributable exclusively to prolonged residence time were excluded. Similar results were obtained with 11C-palmitate. The myocardial avidity for 11C-palmitate was demonstrable by rectilinear whole body scanning in dogs given 5 mCi of the agent intravenously. Diminished 11C-palmitate uptake in zones of myocardium rendered ischemic for 20 minutes prior to reflow in intact dogs was delineated by electrocardiographically gated positron-emission transaxial computer reconstruction tomography. Thus, diminished 11C-FFA extraction, externally detectable, accompanies decreased perfusion in isolated perfused hearts, and decreased 11C-FFA uptake reflecting myocardial ischemia in vivo can be evaluated noninvasively by positron-emission transaxial tomography.

The effect of beta+ range on spatial resolution of imaging systems employing the detection of 511-keV annihilation radiation was determined by measuring the variation in the line-spread functions (LSFs) of positron-emitting radionuclides of 64Cu, 11C, and 15O as compared with the 514-keV gamma-ray emitter 85Sr. These radionuclides have maximum beta+ energies of 0.656, 0.960, and 1.72 MeV, respectively. The LSFs were measured in a tissue-equivalent phantom with high-resolution (approximately 2.4 mm FWHM) and low-resolution (approximately 8.8 mm FWHM) straightbore collimators coupled to a NaI(Tl) detector. Theoretical LSFs for the beta+ ranges were also calculated and convolved with the 85Sr LSF to yield the predicted LSFs for 11C and 15O. The high-resolution study showed a 0% and 2.3% increase in the full-width half-maximum (FWHM) and full-width tenth-maximum (FWO.1M) for the low-energy beta+ of 64Cu and a 37% (FWHM) and 52% (FWO.1M) increase for the high energy beta+ of 15O as compared with 85Sr. However, when the system resolution was decreased to 8.8 mm FWHM, the 64Cu showed no change at FWHM or FWO.1M and the 15O showed a 2.3% (FWHM) and 7.8% (FWO.1M) relative to 85Sr. The predicted LSFs were in good agreement with the experimental. These data indicate that the effect of beta+ range on spatial resolution is minimal unless the beta+ energy is larger than or equal to 1.5 MeV and the system resolution is on the order of a few millimeters.

The NeuroECAT, a multiplane positron tomograph for imaging the brain, was characterized in terms of both quantitative performance and image quality. The tomograph has four modes of operation, defined by the placement of interplane septa and shadow shields. Each mode was fully characterized by measurement of image resolution, axial resolution, resolution uniformity, scatter, accidentals, and deadtime. Each measurement was performed with scattering media simulating the human head, and resolutions were obtained from images processed with reconstruction techniques actually used in patient imaging. The results for the most frequent mode of operation are: image resolution 9.8 +/- 0.2 mm (FWHM), axial resolution 12.4 +/- 0.4 mm, and scatter 8.1% +/- 0.6. At a count rate of 10,000 cps per image plane, accidentals are 9% and the deadtime 3%. Accidentals are measured and subtracted in hardware, and corrections for deadtime loss are calculated from the on-line measurement of triple-coincidence events. Scatter is estimated from the scan data and subtracted in software. Image quality is demonstrated by phantom studies and by the patient images obtained with [F-18] fluorodeoxyglucose and carbon-11 monoxide. The FDG images show clear delineation of the convolutions of the cortical ribbon, internal gray nuclei, internal and external capsules, and other substructures of the brain. The carbon monoxide images, in addition to visualizing the large vessels, clearly show the blood volumes of the cortex, the Sylvian fissure, and the circle of Willis.

An in vivo measurement technique using 15O water and positron CT for quantitation of myocardial blood flow (MBF) was investigated. A closed-chest dog model and NeuroECAT scanner were used in the study. The in vivo technique involves i.v. infusion of 15O water for a duration of 2-3 min. Oxygen-15 water radioactivity in myocardium was imaged with a NeuroECAT scanner for 10 min, starting at the time of tracer infusion. A separate scan following inhalation of 15O CO was obtained to label the blood pool and to help remove the contribution of radioactivity in the blood pool during the 15O water scans. The integrated projection technique was used for calculating MBF. The quantitative microsphere technique for measurement of MBF was performed along with the 15O water study to provide reference values, with which the MBF values by the in vivo technique was compared. Results of 12 experimental runs (in seven animals) show the in vivo technique with 15O water and positron CT can give quantitative flow images of myocardium. The in vivo positron CT measurement was found to correlate well (r = 0.93) with the in vitro values (by microspheres) over the flow range of 40 to 150 ml/min/100 g.

The ECAT was designed and developed as a positron imaging system capable of providing high contrast, high resolution, quantitative images in two-dimensional (2-D) and tomographic formats. The flexibility in its variety of imaging problems. High (HR), medium (MR), and low (LR) tomographic resolutions are 0.95 +/- 0.1, 1.3 +/- 0.1, and 1.7 +/- 0.1 cm FWHM; high, medium, and low resolutions in 2-D images are 0.85 +/- 0.1, 1.3 +/- 0.1 and 1.7 +/- 0.1, depending on resolution mode employed. ECT system efficiency is 30,100, 15,900, and 9,200 c/sec/muCi/cc with a 20-cm diameter phantom at LR, MR, and HR. Because of the geometric, detector, electronic and shielding design of the system, count-rate capability and linearity are high, with minimum detection of scattered radiation and random coincidence. Measured error agrees well with theoretical statistical predictions down to a level of 1.4% standard deviation. The redundant sampling scheme of this system significantly reduces errors caused by motion and detector instability. Scan times are variable from 10 sec to several min/slice and multiple levels are automatically performed by computer control of patient bed. A variety of human studies illustrate image quality, resolution, and efficiency of both ECT and 2-D imaging mode. Examples of the noninvasive study method have been made possible through development of ECT.

A study was carried out to investigate the use of annihilation coincidence detection (ACD) in emmision transaxial reconstruction tomography. The ACD was evaluated in terms of spatial resolution and sensitivity with depth, detection efficiency, effect of pulse-height analysis on resolution and efficiency, correction for attenuation, and cold spot contrast. A prototype positron emission transaxial tomograph (PETT) consisting of a hexagonal array of 24 Nal (Tl) detectors employing ACD was constructed. A fast Fourier transform algorithm was employed to generate the reconstructed image. Computer simulations and phantom and animal studies were carried out to demonstrate that this approach yields tomographic radionuclide images that have high resolution and contrast (hot and cold spot) and that are independent of activity above and below the plane examined. The ACD yields a quantitative nuclear medicine imaging device with high detection efficiency. Comparisons are presented between the ACD and the scintillation camera and scanner. Discussion of the possible applications of the PETT in nuclear medicine is included.

A new method for attenuation correction in positron computed tomography (PCT) has been developed, and it can improve the quality of PCT images. The method requires a short transmission scan by the PCT system. Then boundaries between tissues with significantly different attenuation coefficients are determined from the transmission image by edge-finding techniques. Attenuation correction factors(ACF) are then calculated using these boundaries and the average attenuation coefficients within the enclosed regions. The method has been tested on computer-simulated data, on scans of phantoms, and on patient studies, and has been found effective in reducing the random noise in transmission measurements and in providing more accurate ACFs than the method using geometric attenuation correction. As a result, transmission scan times can be shortened, inconvenience to patients is reduced, and PCT images are improved.

A two-dimensional array detector system, consisting of 4x8 matrices of bismuth germanate (BGO) crystals coupled to four photomultipliers, was evaluated. Coincidence timing resolution of a pair of array detectors was 6.1 ns FWHM and 11.3 ns FWTM. Energy resolution per individual crystal ranged from 16.8 to 24.1% FWHM at 511 keV for an amplitude variation of a factor of 2.8. The signals from the photomultipliers were summed pairwise, the pairs in each dimension subtracted, and the differences divided by the amplitude of the signal summed from all four photomultipliers. The resulting two-pulse-height spectra contained peaks corresponding to the row or column containing the detector of interaction. Six conditions of detector identification were tested. The correct identification of detector ranged from 76% to 87% of the events. The largest single factor in improving crystal identification and spatial resolution was energy discrimination.

A whole-body positron-emission transaxial tomograph (PETT III) was used to image the cross-sectional distribution of 13NH3 and 11CO-hemoglobin in the human brain and heart. Carotid and intravenous bolus injections of 13NH3 in the rhesus monkey had shown that 13NH3 is efficiently extracted by the brain and clears from it slowly (half-time, 40-50 min for carotid injections and 60-70 min for intravenous injections). The intravenous tomographic images in humans showed an excellent relationship between 13NH3 uptakes in the cortex, subcortical white matter, cerebellum, and brain stem and normal blood perfusion or flow in these structures. Cerebral lesions with high (metastasis) and low (stroke) blood flows showed correspondingly high and low uptakes of 13NH3. Large- and small-vascular structures of the brain were also clearly seen in 11CO-hemoglobin tomographic images. Normal myocardium and the ventricular chambers were well defined, and a transmural anterior myocardial infarct was clearly shown. The effective combination of positron transaxial tomography and compounds labeled with positron-emitters provides a safe new method for quantitatively imaging hemodynamic and physiologic functions of selected organs with good tomographic image quality.

When partially purified Eastern equine encephalitis (EEE) virus was centrifuged to equilibrium in CsCl, three virus specific bands were observed. A hemagglutinin was detected at a buoyant density of 1.18 g/cm(3). Infectious EEE virus banded in two positions; most of the virus banded at 1.20 g/cm(3) and a lesser amount banded at 1.22 to 1.23 g/cm(3). Analysis of radioactive profiles of CsCl-fractionated EEE virus labeled with either (32)PO(4) or (3)H-uridine suggested that the hemagglutinin was stripped from the intact EEE virion. The viral origin of the hemagglutinin was verified by inhibition with specific antiserum. Attempts to differentiate between infectious EEE virus of the different buoyant densities showed that the denser particle was neither a virus contaminant nor a density mutant. No evidence was obtained to indicate that the denser particle was an immature form of EEE virus. The two infectious EEE species obtained after CsCl fractionation were indistinguishable antigenically. Furthermore, unfractionated as well as CsCl-fractionated EEE virus sedimented at about 260S in sucrose gradients. These results together with the results of rebanding experiments suggested that the denser EEE species (1.23 g/cm(3)) results from a salt (CsCl)-induced alteration or breakdown of the EEE virion (1.20 g/cm(3)), and that it arises as the hemagglutinin is stripped from the surface of the EEE virion.