Alagille syndrome (AGS) is caused by mutations in the gene for the Notch signaling pathway ligand Jagged1 (JAG1), which are found in 94% of patients. To identify the cause of disease in patients without JAG1 mutations, we screened 11 JAG1 mutation-negative probands with AGS for alterations in the gene for the Notch2 receptor (NOTCH2). We found NOTCH2 mutations segregating in two families and identified five affected individuals. Renal manifestations, a minor feature in AGS, were present in all the affected individuals. This demonstrates that AGS is a heterogeneous disorder and implicates NOTCH2 mutations in human disease.

Statistics. All data were subjected to statistical analysis in GraphPad Prism 6. When WT and transgene GCG-Gq DREADD mice were compared, 2-tailed unpaired t tests were performed. Time course tests were analyzed with 2-way repeated measures ANOVA with treatment and time points as independent variables. When the same mice received alternating CNO and saline treatments, paired 2-tailed t tests were used to evaluate data for significance. Data from once-performed behavioral tests, such as the open field and elevated plus maze, were analyzed with unpaired 2-tailed t tests. Data are expressed as mean ± SEM. Statistical significance was considered with P < 0.05.Study approval. All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Virginia and conducted in accordance with its guidelines.

Alagille syndrome (AGS) is caused by heterozygous mutations in JAG1, and mutations have been previously reported in about 70% of patients who meet clinical diagnostic criteria. We studied a cohort of 247 clinically well-defined patients, and using an aggressive and sequential screening approach we identified JAG1 mutations in 94% of individuals. Mutations were found in 232 out of 247 patients studied and 83 of the mutations were novel. This increase in the mutation rate was accomplished by combining rigorous clinical phenotyping, with a combination of mutation detection techniques, including fluorescence in situ hybridization (FISH), genomic and cDNA sequencing, and quantitative PCR. This higher rate of mutation identification has implications for clinical practice, facilitating genetic counseling, prenatal diagnosis, and evaluation of living-related liver transplant donors. Our results suggest that more aggressive screening may similarly increase the rate of mutation detection in other dominant and recessive disorders.

The use of array technology to define chromosome deletions and duplications is bringing us closer to establishing a genotype/phenotype map of genomic copy number alterations. We studied 21 patients and 5 relatives with deletions of the short arm of chromosome 20 using the Illumina HumanHap550 SNP array to 1) more accurately determine the deletion sizes, 2) identify and compare breakpoints, 3) establish genotype/phenotype correlations and 4) investigate the use of the HumanHap550 platform for analysis of chromosome deletions. Deletions ranged from 95kb to 14.62Mb, and all of the breakpoints were unique. Eleven patients had deletions between 95kb and 4Mb and these individuals had normal development, with no anomalies outside of those associated with Alagille syndrome. The proximal and distal boundaries of these eleven deletions constitute a 5.4MB region, and we propose that haploinsufficiency for only 1 of the 12 genes in this region causes phenotypic abnormalities. This defines the JAG1 associated critical region, in which deletions do not confer findings other than those associated with Alagille syndrome. The other 10 patients had deletions between 3.28Mb and 14.62Mb, which extended outside the critical region, and notably, all of these patients, had developmental delay. This group had other findings such as autism, scoliosis and bifid uvula. We identified 47 additional polymorphic genome-wide copy number variants (>20 SNPs), with 0-5 variants called per patient. Deletions of the short arm of chromosome 20 are associated with relatively mild and limited clinical anomalies. The use of SNP arrays provides accurate high-resolution definition of genomic abnormalities.

The ability to learn, remember, and respond to emotional events is a powerful survival strategy. However, dysregulated behavioral and physiological responses to these memories are maladaptive. To fully understand learned fear and the pathologies that arise during response malfunction we must reveal the environmental variables that influence learned fear responses. Light, a ubiquitous environmental feature, modulates cognition and anxiety. We hypothesized that light modulates responses to learned fear. Using tone-cued fear conditioning, we found that light enhances behavioral responses to learned fear in C57BL/6J mice. Mice in light freeze more in response to a conditioned cue than mice in darkness. The absence of significant freezing during a 2-wk habituation period and during intertrial intervals indicated that light specifically modulates freezing to the learned acoustic cue rather than the context of the experimental chamber. Repeating our assay in two photoreceptor mutant models, Pde6b rd1/rd1 and Opn4 −/− mice, revealed that lightdependent enhancement of conditioned fear is driven primarily by the rods and/or cones. By repeating our protocol with an altered lighting regimen, we found that lighting conditions acutely modulate responses when altered between conditioning and testing. This is manifested either as an enhancement of freezing when light is added during testing or as a depression of freezing when light is removed during testing. Acute enhancement, but not depression, requires both rod/cone-and melanopsin-dependent photoreception. Our results demonstrate a modulation by light of behavioral responses to learned fear.L ight is a pervasive feature of the environment and exerts broad effects on behavior and physiology via two parallel pathways (1). The familiar image-forming visual pathway allows discernment of objects in the environment according to physical qualities: their color, form, texture, and motion. The parallel non-image forming (NIF) pathway enables light to exert numerous effects on physiology and behavior independently of image formation, such as pupil constriction, modulation of heart rate, and the synchronization of circadian rhythms and sleepwake cycles to the daily light-dark cycle (2). In addition to the effects of light on basic physiological functions, light also modulates higher-order cognitive processes, including anxiety, mood, and alertness/awakeness (3, 4). The retina, the sole photosensory organ in mammals, projects directly to brain regions involved in emotional responses. Among these are the amygdala, the bed nucleus of the stria terminalis, and the periaqueductal gray (5). Activity in some of these regions is known to be acutely modulated by light in a wavelength-dependent manner (3, 6), whereas the link between photoreception and function in other retinorecipient emotional processing areas remains to be elucidated.Brain sites involved in emotional processing participate in the critical function of learning and remembering emotionally arousing events. This function enables ...

The medial prefrontal cortex (mPFC) is implicated in aspects of executive function, that include the modulation of attentional and memory processes involved in goal selection. Food-seeking behavior has been shown to involve activation of the mPFC, both during the execution of strategies designed to obtain food and during the consumption of food itself. As these behaviors likely require differential engagement of the prefrontal cortex, we hypothesized that the pattern of neuronal activation would also be behavior dependent. In this study we describe, for the first time, the expression of Fos in different layers and cell types of the infralimbic/dorsal peduncular and prelimbic/anterior cingulate subdivisions of mouse mPFC following both the consumption of palatable food and following exploratory activity of the animal directed at obtaining food reward. While both manipulations led to increases of Fos expression in principal excitatory neurons relative to control, food-directed exploratory activity produced a significantly greater increase in Fos expression than observed in the food intake condition. Consequently, we hypothesized that mPFC interneuron activation would also be differentially engaged by these manipulations. Interestingly, Fos expression patterns differed substantially between treatments and interneuron subtype, illustrating how the differential engagement of subsets of mPFC interneurons depends on the behavioral state. In our experiments, both vasoactive intestinal peptide- and parvalbumin-expressing neurons showed enhanced Fos expression only during the food-dependent exploratory task and not during food intake. Conversely, elevations in arcuate and paraventricular hypothalamic fos expression were only observed following food intake and not following food driven exploration. Our data suggest that select activation of these cell types may be required to support high cognitive demand states such as observed during exploration while being dispensable during the ingestion of freely available food.

The medial prefrontal cortex (mPFC) is involved in a wide range of executive cognitive functions, including reward evaluation, decision-making, memory extinction, mood, and task switching. Manipulation of the mPFC has been shown to alter food intake and food reward valuation, but whether exclusive stimulation of mPFC pyramidal neurons (PN), which form the principle output of the mPFC, is sufficient to mediate food rewarded instrumental behavior is unknown. We sought to determine the behavioral consequences of manipulating mPFC output by exciting PN in mouse mPFC during performance of a panel of behavioral assays, focusing on food reward. We found that increasing mPFC pyramidal cell output using designer receptors exclusively activated by designer drugs (DREADD) enhanced performance in instrumental food reward assays that assess food seeking behavior, while sparing effects on affect and food intake. Specifically, activation of mPFC PN enhanced operant responding for food reward, reinstatement of palatable food seeking, and suppression of impulsive responding for food reward. Conversely, activation of mPFC PN had no effect on unconditioned food intake, social interaction, or behavior in an open field. Furthermore, we found that behavioral outcome is influenced by the degree of mPFC activation, with a low drive sufficient to enhance operant responding and a higher drive required to alter impulsivity. Additionally, we provide data demonstrating that DREADD stimulation involves a nitric oxide (NO) synthase dependent pathway, similar to endogenous muscarinic M3 receptor stimulation, a finding that provides novel mechanistic insight into an increasingly widespread method of remote neuronal control.

Melanopsin (gene symbol : Opn4) is the G protein-coupled photopigment that confers photosensitivity upon intrinsically photosensitive retinal ganglion cells (ipRGCs). ipRGCs are the third class of retinal photoreceptor in mammals, complementing the two previously identified classes, the rods and cones. This novel class, however, differs from rods and cones in many significant ways. First, ipRGCs are more similar morphologically to other retinal ganglion cell classes than to other retinal photoreceptors, i.e., rods and cones. Instead of having photopigment concentrated in a specialized light-absorbing cellular domain such as the outer segment, ipRGCs have photopigment distributed throughout the plasma membrane of the cell. Second, the phototransduction cascade of ipRGCs more closely resembles that of the rhabdomeric photoreceptors that are typically found in the invertebrates rather than that of ciliary photoreceptors typical of vertebrate visual systems. Accordingly, like the rhabdomeric photoreceptors of invertebrates, ipRGCs depolarize in response to illumination while rods and cones hyperpolarize. Third, in addition to their inherent light sensitivity, ipRGCs also function as a conduit for information that originates in the rods and cones and is conveyed to the brain for the purposes of generating non-visual light responses.