Publications by authors named "Patrick M Nolan"

65 Publications

Zfhx3-mediated genetic ablation of the SCN abolishes light entrainable circadian activity while sparing food anticipatory activity.

iScience 2021 Oct 16;24(10):103142. Epub 2021 Sep 16.

MRC Harwell Institute, Harwell Science Campus, Oxfordshire OX11 0RD, UK.

Circadian rhythms persist in almost all organisms and are crucial for maintaining appropriate timing in physiology and behaviour. Here, we describe a mouse mutant where the central mammalian pacemaker, the suprachiasmatic nucleus (SCN), has been genetically ablated by conditional deletion of the transcription factor in the developing hypothalamus. Mutants were arrhythmic over the light-dark cycle and in constant darkness. Moreover, rhythms of metabolic parameters were ablated although molecular oscillations in the liver maintained some rhythmicity. Despite disruptions to SCN cell identity and circuitry, mutants could still anticipate food availability, yet other zeitgebers - including social cues from cage-mates - were ineffective in restoring rhythmicity although activity levels in mutants were altered. This work highlights a critical role for in the development of a functional SCN, while its genetic ablation further defines the contribution of SCN circuitry in orchestrating physiological and behavioral responses to environmental signals.
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http://dx.doi.org/10.1016/j.isci.2021.103142DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8487057PMC
October 2021

Comprehensive phenotypic analysis of the Dp1Tyb mouse strain reveals a broad range of Down syndrome-related phenotypes.

Dis Model Mech 2021 Oct 15;14(10). Epub 2021 Oct 15.

The Francis Crick Institute, London NW1 1AT, UK.

Down syndrome (DS), trisomy 21, results in many complex phenotypes including cognitive deficits, heart defects and craniofacial alterations. Phenotypes arise from an extra copy of human chromosome 21 (Hsa21) genes. However, these dosage-sensitive causative genes remain unknown. Animal models enable identification of genes and pathological mechanisms. The Dp1Tyb mouse model of DS has an extra copy of 63% of Hsa21-orthologous mouse genes. In order to establish whether this model recapitulates DS phenotypes, we comprehensively phenotyped Dp1Tyb mice using 28 tests of different physiological systems and found that 468 out of 1800 parameters were significantly altered. We show that Dp1Tyb mice have wide-ranging DS-like phenotypes, including aberrant erythropoiesis and megakaryopoiesis, reduced bone density, craniofacial changes, altered cardiac function, a pre-diabetic state, and deficits in memory, locomotion, hearing and sleep. Thus, Dp1Tyb mice are an excellent model for investigating complex DS phenotype-genotype relationships for this common disorder.
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http://dx.doi.org/10.1242/dmm.049157DOI Listing
October 2021

Zfhx3 modulates retinal sensitivity and circadian responses to light.

FASEB J 2021 09;35(9):e21802

MRC Harwell Institute, Oxfordshire, UK.

Mutations in transcription factors often exhibit pleiotropic effects related to their complex expression patterns and multiple regulatory targets. One such mutation in the zinc finger homeobox 3 (ZFHX3) transcription factor, short circuit (Sci, Zfhx3 ), is associated with significant circadian deficits in mice. However, given evidence of its retinal expression, we set out to establish the effects of the mutation on retinal function using molecular, cellular, behavioral and electrophysiological measures. Immunohistochemistry confirms the expression of ZFHX3 in multiple retinal cell types, including GABAergic amacrine cells and retinal ganglion cells including intrinsically photosensitive retinal ganglion cells (ipRGCs). Zfhx3 mutants display reduced light responsiveness in locomotor activity and circadian entrainment, relatively normal electroretinogram and optomotor responses but exhibit an unexpected pupillary reflex phenotype with markedly increased sensitivity. Furthermore, multiple electrode array recordings of Zfhx3 retina show an increased sensitivity of ipRGC light responses.
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http://dx.doi.org/10.1096/fj.202100563RDOI Listing
September 2021

Simultaneous Assessment of Circadian Rhythms and Sleep in Mice Using Passive Infrared Sensors: A User's Guide.

Curr Protoc Mouse Biol 2020 Sep;10(3):e81

Sleep and Circadian Neuroscience Institute (SCNi), Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, UK.

The 24-hr cycle of activity and sleep provides perhaps the most familiar example of circadian rhythms. In mammals, circadian activity rhythms are generated by a master biological clock located in the hypothalamic suprachiasmatic nuclei (SCN). This clock is synchronized (entrained) to the external light environment via light input from retinal photoreceptors. However, sleep is not a simple circadian output and also is regulated by a homeostatic process whereby extended wakefulness increases the need for subsequent sleep. As such, the amount and distribution of sleep depends upon the interaction between both circadian and homeostatic processes. Moreover, the study of circadian activity and sleep is not confined only to these specialized fields. Sleep and circadian rhythm disruption is common in many conditions, ranging from neurological and metabolic disorders to aging. Such disruption is associated with a range of negative consequences including cognitive impairment and mood disorders, as well as immune and metabolic dysfunction. As circadian activity and sleep are hallmarks of normal healthy physiology, they also provide valuable welfare indicators. However, traditional methods for the monitoring of circadian rhythms and sleep in mice can require separate specialized resources as well as significant expertise. Here, we outline a low-cost, non-invasive, and open-source method for the simultaneous assessment of circadian activity and sleep in mice. This protocol describes both the assembly of the hardware used and the capture and analysis of data without the need for expertise in electronics or data processing. © 2020 Wiley Periodicals LLC. Basic Protocol: Assembly of a PIR system for basic activity and sleep recordings Alternate Protocol: Data collection using Raspberry Pi Support Protocol: Circadian analysis using PIR sensors.
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http://dx.doi.org/10.1002/cpmo.81DOI Listing
September 2020

Forward genetics identifies a novel sleep mutant with sleep state inertia and REM sleep deficits.

Sci Adv 2020 Aug 12;6(33):eabb3567. Epub 2020 Aug 12.

Mammalian Genetics Unit, MRC Harwell Institute, Harwell Science and Innovation Campus, Oxfordshire, UK.

Switches between global sleep and wakefulness states are believed to be dictated by top-down influences arising from subcortical nuclei. Using forward genetics and in vivo electrophysiology, we identified a recessive mouse mutant line characterized by a substantially reduced propensity to transition between wake and sleep states with an especially pronounced deficit in initiating rapid eye movement (REM) sleep episodes. The causative mutation, an Ile102Asn substitution in the synaptic vesicular protein, VAMP2, was associated with morphological synaptic changes and specific behavioral deficits, while in vitro electrophysiological investigations with fluorescence imaging revealed a markedly diminished probability of vesicular release in mutants. Our data show that global shifts in the synaptic efficiency across brain-wide networks leads to an altered probability of vigilance state transitions, possibly as a result of an altered excitability balance within local circuits controlling sleep-wake architecture.
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http://dx.doi.org/10.1126/sciadv.abb3567DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7423362PMC
August 2020

Phenotyping in Mice Using Continuous Home Cage Monitoring and Ultrasonic Vocalization Recordings.

Curr Protoc Mouse Biol 2020 Sep;10(3):e80

Medical Research Council Harwell Institute, Harwell, Oxfordshire, United Kingdom.

Over the last century, the study of mouse behavior has uncovered insights into brain molecular mechanisms while revealing potential causes of many neurological disorders. To this end, researchers have widely exploited the use of mutant strains, including those generated in mutagenesis screens and those produced using increasingly sophisticated genome engineering technologies. It is now relatively easy to access mouse models carrying alleles that faithfully recapitulate changes found in human patients or bearing variants of genes that provide data on those genes' functions. Concurrent with these developments has been an appreciation of the limitations of some current testing platforms, especially those monitoring complex behaviors. Out-of-cage observational testing is useful in describing overt persistent phenotypes but risks missing sporadic or intermittent events. Furthermore, measuring the progression of a phenotype, potentially over many months, can be difficult while relying on assays that may be susceptible to changes in the testing environment. In recent years, there has also been increasing awareness that measurement of behaviors in isolation can be limiting, given that mice attempt to hide behavioral cues of vulnerability. To overcome these limitations, laboratory animal science is capitalizing on progress in data capture and processing expertise. Moreover, as additional recording modes become commonplace, ultrasonic vocalization recording is an appealing focus, as mice use vocalizations in various social contexts. Using video and audio technologies, we record the voluntary, unprovoked behaviors and vocalizations of mice in social groups. Adoption of these approaches is undoubtedly set to increase, as they capture the round-the-clock behavior of mouse strains. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Continuous recording of home cage activity using the Home Cage Analyzer (HCA) system Support Protocol: Subcutaneous insertion of a radio frequency identification microchip in the inguinal area Basic Protocol 2: Continuous recording of mouse ultrasonic vocalizations in the home cage.
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http://dx.doi.org/10.1002/cpmo.80DOI Listing
September 2020

Loss of Bardet-Biedl syndrome proteins causes synaptic aberrations in principal neurons.

PLoS Biol 2019 09 3;17(9):e3000414. Epub 2019 Sep 3.

Great Ormond Street Institute of Child Health, University College London, London, United Kingdom.

Bardet-Biedl syndrome (BBS), a ciliopathy, is a rare genetic condition characterised by retinal degeneration, obesity, kidney failure, and cognitive impairment. In spite of progress made in our general understanding of BBS aetiology, the molecular and cellular mechanisms underlying cognitive impairment in BBS remain elusive. Here, we report that the loss of BBS proteins causes synaptic dysfunction in principal neurons, providing a possible explanation for the cognitive impairment phenotype observed in BBS patients. Using synaptosomal proteomics and immunocytochemistry, we demonstrate the presence of Bbs proteins in the postsynaptic density (PSD) of hippocampal neurons. Loss of Bbs results in a significant reduction of dendritic spines in principal neurons of Bbs mouse models. Furthermore, we show that spine deficiency correlates with events that destabilise spine architecture, such as impaired spine membrane receptor signalling, known to be involved in the maintenance of dendritic spines. Our findings suggest a role for BBS proteins in dendritic spine homeostasis that may be linked to the cognitive phenotype observed in BBS.
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http://dx.doi.org/10.1371/journal.pbio.3000414DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6743795PMC
September 2019

The guanine nucleotide exchange factor, Spata13, influences social behaviour and nocturnal activity.

Mamm Genome 2019 04 24;30(3-4):54-62. Epub 2019 Apr 24.

MRC Harwell Institute, Harwell Campus, Didcot, Oxfordshire, OX11 0RD, UK.

Spermatogenesis-associated protein 13 (Spata13) is a guanine nucleotide exchange factor (GEF) enriched in discrete brain regions in the adult, with pronounced expression in the extended central amygdala (CeA). Loss of Spata13, also known as the adenomatous polyposis coli exchange factor Asef2, has no identifiable phenotype although it has been shown to reduce the number and size of intestinal tumours in Apc (Min/+) mice. Nevertheless, its brain-related functions have not been investigated. To pursue this, we have generated a Spata13 knockout mouse line using CRISPR-mediated deletion of an exon containing the GTPase domain that is common to multiple isoforms. Homozygous mutants were viable and appeared normal. We subjected both male and female cohorts to a comprehensive battery of behavioural tests designed to investigate particular CeA-related functions. Here, we show that Spata13 modulates social behaviour with homozygous mutants being subordinate to wildtype controls. Furthermore, female homozygotes show increased activity in home cages during the dark phase of the light-dark cycle. In summary, Spata13 modulates social hierarchy in both male and female mice in addition to affecting voluntary activity in females.
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http://dx.doi.org/10.1007/s00335-019-09800-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6491400PMC
April 2019

Loss of disrupts synaptic AMPA receptor function, and results in neurodevelopmental, motor, cognitive and electrographical abnormalities.

Dis Model Mech 2019 02 22;12(2). Epub 2019 Feb 22.

MRC Harwell Institute, Harwell Campus, Oxfordshire OX11 0RD, UK

Loss-of-function mutations in a human AMPA receptor-associated protein, ferric chelate reductase 1-like (FRRS1L), are associated with a devastating neurological condition incorporating choreoathetosis, cognitive deficits and epileptic encephalopathies. Furthermore, evidence from overexpression and studies has implicated FRRS1L in AMPA receptor biogenesis, suggesting that changes in glutamatergic signalling might underlie the disorder. Here, we investigated the neurological and neurobehavioural correlates of the disorder using a mouse null mutant. The study revealed several neurological defects that mirrored those seen in human patients. We established that mice lacking suffered from a broad spectrum of early-onset motor deficits with no progressive, age-related deterioration. Moreover, mice were hyperactive, irrespective of test environment, exhibited working memory deficits and displayed significant sleep fragmentation. Longitudinal electroencephalographic (EEG) recordings also revealed abnormal EEG results in mice. Parallel investigations into disease aetiology identified a specific deficiency in AMPA receptor levels in the brain of mice, while the general levels of several other synaptic components remained unchanged, with no obvious alterations in the number of synapses. Furthermore, we established that deletion results in an increased proportion of immature AMPA receptors, indicated by incomplete glycosylation of GLUA2 (also known as GRIA2) and GLUA4 (also known as GRIA4) AMPA receptor proteins. This incomplete maturation leads to cytoplasmic retention and a reduction of those specific AMPA receptor levels in the postsynaptic membrane. Overall, this study determines, for the first time , how loss of FRRS1L function can affect glutamatergic signalling, and provides mechanistic insight into the development and progression of a human hyperkinetic disorder.This article has an associated First Person interview with the first author of the paper.
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http://dx.doi.org/10.1242/dmm.036806DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6398485PMC
February 2019

Differential roles for cryptochromes in the mammalian retinal clock.

FASEB J 2018 08 21;32(8):4302-4314. Epub 2018 Mar 21.

Sleep and Circadian Neuroscience Institute, University of Oxford, Oxford, United Kingdom.

Cryptochromes 1 and 2 (CRY1/2) are key components of the negative limb of the mammalian circadian clock. Like many peripheral tissues, Cry1 and -2 are expressed in the retina, where they are thought to play a role in regulating rhythmic physiology. However, studies differ in consensus as to their localization and function, and CRY1 immunostaining has not been convincingly demonstrated in the retina. Here we describe the expression and function of CRY1 and -2 in the mouse retina in both sexes. Unexpectedly, we show that CRY1 is expressed throughout all retinal layers, whereas CRY2 is restricted to the photoreceptor layer. Retinal period 2::luciferase recordings from CRY1-deficient mice show reduced clock robustness and stability, while those from CRY2-deficient mice show normal, albeit long-period, rhythms. In functional studies, we then investigated well-defined rhythms in retinal physiology. Rhythms in the photopic electroretinogram, contrast sensitivity, and pupillary light response were all severely attenuated or abolished in CRY1-deficient mice. In contrast, these physiological rhythms are largely unaffected in mice lacking CRY2, and only photopic electroretinogram rhythms are affected. Together, our data suggest that CRY1 is an essential component of the mammalian retinal clock, whereas CRY2 has a more limited role.-Wong, J. C. Y., Smyllie, N. J., Banks, G. T., Pothecary, C. A., Barnard, A. R., Maywood, E. S., Jagannath, A., Hughes, S., van der Horst, G. T. J., MacLaren, R. E., Hankins, M. W., Hastings, M. H., Nolan, P. M., Foster, R. G., Peirson, S. N. Differential roles for cryptochromes in the mammalian retinal clock.
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http://dx.doi.org/10.1096/fj.201701165RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6071063PMC
August 2018

The after-hours circadian mutant has reduced phenotypic plasticity in behaviors at multiple timescales and in sleep homeostasis.

Sci Rep 2017 12 19;7(1):17765. Epub 2017 Dec 19.

Department of Neuroscience and Brain Technologies - Genetics and Epigenetics of Behaviour - Istituto Italiano di Tecnologia, via Morego, 30, 16163, Genova, Italy.

Circadian clock is known to adapt to environmental changes and can significantly influence cognitive and physiological functions. In this work, we report specific behavioral, cognitive, and sleep homeostatic defects in the after hours (Afh) circadian mouse mutant, which is characterized by lengthened circadian period. We found that the circadian timing irregularities in Afh mice resulted in higher interval timing uncertainty and suboptimal decisions due to incapability of processing probabilities. Our phenotypic observations further suggested that Afh mutants failed to exhibit the necessary phenotypic plasticity for adapting to temporal changes at multiple time scales (seconds-to-minutes to circadian). These behavioral effects of Afh mutation were complemented by the specific disruption of the Per/Cry circadian regulatory complex in brain regions that govern food anticipatory behaviors, sleep, and timing. We derive statistical predictions, which indicate that circadian clock and sleep are complementary processes in controlling behavioral/cognitive performance during 24 hrs. The results of this study have pivotal implications for understanding how the circadian clock modulates sleep and behavior.
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http://dx.doi.org/10.1038/s41598-017-18130-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5736711PMC
December 2017

Disruption of the homeodomain transcription factor orthopedia homeobox (Otp) is associated with obesity and anxiety.

Mol Metab 2017 11 24;6(11):1419-1428. Epub 2017 Aug 24.

MRC Harwell Institute, Mammalian Genetics Unit and Mary Lyon Centre, Harwell Campus, Oxfordshire, OX11 0RD, UK. Electronic address:

Objective: Genetic studies in obese rodents and humans can provide novel insights into the mechanisms involved in energy homeostasis.

Methods: In this study, we genetically mapped the chromosomal region underlying the development of severe obesity in a mouse line identified as part of a dominant N-ethyl-N-nitrosourea (ENU) mutagenesis screen. We characterized the metabolic and behavioral phenotype of obese mutant mice and examined changes in hypothalamic gene expression. In humans, we examined genetic data from people with severe early onset obesity.

Results: We identified an obese mouse heterozygous for a missense mutation (pR108W) in orthopedia homeobox (Otp), a homeodomain containing transcription factor required for the development of neuroendocrine cell lineages in the hypothalamus, a region of the brain important in the regulation of energy homeostasis. Otp mice exhibit increased food intake, weight gain, and anxiety when in novel environments or singly housed, phenotypes that may be partially explained by reduced hypothalamic expression of oxytocin and arginine vasopressin. R108W affects the highly conserved homeodomain, impairs DNA binding, and alters transcriptional activity in cells. We sequenced OTP in 2548 people with severe early-onset obesity and found a rare heterozygous loss of function variant in the homeodomain (Q153R) in a patient who also had features of attention deficit disorder.

Conclusions: OTP is involved in mammalian energy homeostasis and behavior and appears to be necessary for the development of hypothalamic neural circuits. Further studies will be needed to investigate the contribution of rare variants in OTP to human energy homeostasis.
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http://dx.doi.org/10.1016/j.molmet.2017.08.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5681237PMC
November 2017

Meta-analysis of transcriptomic datasets identifies genes enriched in the mammalian circadian pacemaker.

Nucleic Acids Res 2017 Sep;45(17):9860-9873

Sleep and Circadian Neuroscience Institute (SCNi), Nuffield Department of Clinical Neurosciences, University of Oxford, Oxford, OX1 3RE, UK.

The master circadian pacemaker in mammals is located in the suprachiasmatic nuclei (SCN) which regulate physiology and behaviour, as well as coordinating peripheral clocks throughout the body. Investigating the function of the SCN has often focused on the identification of rhythmically expressed genes. However, not all genes critical for SCN function are rhythmically expressed. An alternative strategy is to characterize those genes that are selectively enriched in the SCN. Here, we examined the transcriptome of the SCN and whole brain (WB) of mice using meta-analysis of publicly deposited data across a range of microarray platforms and RNA-Seq data. A total of 79 microarrays were used (24 SCN and 55 WB samples, 4 different microarray platforms), alongside 17 RNA-Seq data files (7 SCN and 10 WB). 31 684 MGI gene symbols had data for at least one platform. Meta-analysis using a random effects model for weighting individual effect sizes (derived from differential expression between relevant SCN and WB samples) reliably detected known SCN markers. SCN-enriched transcripts identified in this study provide novel insights into SCN function, including identifying genes which may play key roles in SCN physiology or provide SCN-specific drivers.
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http://dx.doi.org/10.1093/nar/gkx714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5737434PMC
September 2017

Inducible Knockout of Mouse Zfhx3 Emphasizes Its Key Role in Setting the Pace and Amplitude of the Adult Circadian Clock.

J Biol Rhythms 2017 Oct 17;32(5):433-443. Epub 2017 Aug 17.

1 Vertex Pharmaceuticals, Boston, MA 02210.

The transcription factor zinc finger homeobox 3 (ZFHX3) plays a key role in coupling intracellular transcriptional-translational oscillations with intercellular synchrony in mouse suprachiasmatic nucleus (SCN). However, like many key players in central nervous system function, ZFHX3 serves an important role in neurulation and neuronal terminal differentiation while retaining discrete additional functions in the adult SCN. Recently, using a dominant missense mutation in mouse Zfhx3, we established that this gene can modify circadian period and sleep in adult animals. Nevertheless, we were still concerned that the neurodevelopmental consequences of ZFHX3 dysfunction in this mutant may interfere with, or confound, its critical adult-specific roles in SCN circadian function. To circumvent the developmental consequences of Zfhx3 deletion, we crossed a conditional null Zfhx3 mutant to an inducible, ubiquitously expressed Cre line (B6.Cg-Tg(UBC-cre/ERT2)1Ejb/J). This enabled us to assess circadian behavior in the same adult animals both before and after Cre-mediated excision of the critical Zfhx3 exons using tamoxifen treatment. Remarkably, we found a strong and significant alteration in circadian behavior in tamoxifen-treated homozygous animals with no phenotypic changes in heterozygous or control animals. Cre-mediated excision of Zfhx3 critical exons in adult animals resulted in shortening of the period of wheel-running in constant darkness by more than 1 h in the majority of homozygotes while, in 30% of animals, excision resulted in complete behavioral arrhythmicity. In addition, we found that homozygous animals reentrain almost immediately to 6-h phase advances in the light-dark cycle. No additional overt phenotypic changes were evident in treated homozygous animals. These findings confirm a sustained and significant role for ZFHX3 in maintaining rhythmicity in the adult mammalian circadian system.
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http://dx.doi.org/10.1177/0748730417722631DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5692189PMC
October 2017

Phenotyping first-generation genome editing mutants: a new standard?

Mamm Genome 2017 08 29;28(7-8):377-382. Epub 2017 Jul 29.

Mammalian Genetics Unit, MRC Harwell Institute, Harwell Science Campus, Didcot, Oxfordshire, UK.

The unprecedented efficiency of the CRISPR/Cas9 system in genome engineering has opened the prospect of employing mutant founders for phenotyping cohorts, thus accelerating research projects by circumventing the requirement to generate cohorts using conventional two- or three-generation crosses. However, these first-generation mutants are often genetic mosaics, with a complex and difficult to define genetic make-up. Here, we discuss the potential benefits, challenges and scientific validity of such models.
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http://dx.doi.org/10.1007/s00335-017-9711-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5569115PMC
August 2017

Assessing mouse behaviour throughout the light/dark cycle using automated in-cage analysis tools.

J Neurosci Methods 2018 04 26;300:37-47. Epub 2017 Apr 26.

Mammalian Genetics Unit, MRC Harwell Institute, Harwell Science Campus, Oxfordshire, UK. Electronic address:

An important factor in reducing variability in mouse test outcomes has been to develop assays that can be used for continuous automated home cage assessment. Our experience has shown that this has been most evidenced in long-term assessment of wheel-running activity in mice. Historically, wheel-running in mice and other rodents have been used as a robust assay to determine, with precision, the inherent period of circadian rhythms in mice. Furthermore, this assay has been instrumental in dissecting the molecular genetic basis of mammalian circadian rhythms. In teasing out the elements of this test that have determined its robustness - automated assessment of an unforced behaviour in the home cage over long time intervals - we and others have been investigating whether similar test apparatus could be used to accurately discriminate differences in distinct behavioural parameters in mice. Firstly, using these systems, we explored behaviours in a number of mouse inbred strains to determine whether we could extract biologically meaningful differences. Secondly, we tested a number of relevant mutant lines to determine how discriminative these parameters were. Our findings show that, when compared to conventional out-of-cage phenotyping, a far deeper understanding of mouse mutant phenotype can be established by monitoring behaviour in the home cage over one or more light:dark cycles.
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http://dx.doi.org/10.1016/j.jneumeth.2017.04.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5909039PMC
April 2018

Novel gene function revealed by mouse mutagenesis screens for models of age-related disease.

Nat Commun 2016 08 18;7:12444. Epub 2016 Aug 18.

The Nuffield Laboratory of Ophthalmology &NIHR Oxford Biomedical Research Centre, University of Oxford, Oxford OX3 9DU, UK.

Determining the genetic bases of age-related disease remains a major challenge requiring a spectrum of approaches from human and clinical genetics to the utilization of model organism studies. Here we report a large-scale genetic screen in mice employing a phenotype-driven discovery platform to identify mutations resulting in age-related disease, both late-onset and progressive. We have utilized N-ethyl-N-nitrosourea mutagenesis to generate pedigrees of mutagenized mice that were subject to recurrent screens for mutant phenotypes as the mice aged. In total, we identify 105 distinct mutant lines from 157 pedigrees analysed, out of which 27 are late-onset phenotypes across a range of physiological systems. Using whole-genome sequencing we uncover the underlying genes for 44 of these mutant phenotypes, including 12 late-onset phenotypes. These genes reveal a number of novel pathways involved with age-related disease. We illustrate our findings by the recovery and characterization of a novel mouse model of age-related hearing loss.
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http://dx.doi.org/10.1038/ncomms12444DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4992138PMC
August 2016

Analysis of Individual Mouse Activity in Group Housed Animals of Different Inbred Strains using a Novel Automated Home Cage Analysis System.

Front Behav Neurosci 2016 10;10:106. Epub 2016 Jun 10.

Actual Analytics LtdEdinburgh, UK; School of Informatics, University of EdinburghEdinburgh, UK.

Central nervous system disorders such as autism as well as the range of neurodegenerative diseases such as Huntington's disease are commonly investigated using genetically altered mouse models. The current system for characterizing these mice usually involves removing the animals from their home-cage environment and placing them into novel environments where they undergo a battery of tests measuring a range of behavioral and physical phenotypes. These tests are often only conducted for short periods of times in social isolation. However, human manifestations of such disorders are often characterized by multiple phenotypes, presented over long periods of time and leading to significant social impacts. Here, we have developed a system which will allow the automated monitoring of individual mice housed socially in the cage they are reared and housed in, within established social groups and over long periods of time. We demonstrate that the system accurately reports individual locomotor behavior within the group and that the measurements taken can provide unique insights into the effects of genetic background on individual and group behavior not previously recognized.
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http://dx.doi.org/10.3389/fnbeh.2016.00106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4901040PMC
July 2016

The Zfhx3-Mediated Axis Regulates Sleep and Interval Timing in Mice.

Cell Rep 2016 07 30;16(3):615-21. Epub 2016 Jun 30.

Department of Neuroscience and Brain Technologies, Istituto Italiano di Tecnologia, via Morego, 30, 16163 Genova, Italy. Electronic address:

An AT motif-dependent axis, modulated by the transcription factor Zfhx3, influences the circadian clock in mice. In particular, gain of function of Zfhx3 significantly shortens circadian rhythms and alters the transcriptional activity of an important class of neuropeptides that controls intercellular signaling in the suprachiasmatic nucleus (SCN) of the hypothalamus. The ZFHX3/AT axis revealed an important, largely cell-nonautonomous control of the circadian clock. Here, by studying the recently identified circadian mouse mutant Zfhx3(Sci/+), we identify significant effects on sleep homeostasis, a phenomenon that is outside the canonical circadian clock system and that is modulated by the activity of those neuropeptides at a circuit level. We show that the Zfhx3(Sci/+) mutation accelerates the circadian clock at both the hourly scale (i.e., advancing circadian rhythms) and the seconds-to-minutes scale (i.e., anticipating behavioral responses) in mice. The in vivo results are accompanied by a significant presence of sleep targets among protein-protein interactions of the Zfhx3(Sci/+)-dependent network.
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http://dx.doi.org/10.1016/j.celrep.2016.06.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5991551PMC
July 2016

Melanopsin Regulates Both Sleep-Promoting and Arousal-Promoting Responses to Light.

PLoS Biol 2016 06 8;14(6):e1002482. Epub 2016 Jun 8.

Sleep and Circadian Neuroscience Institute (SCNi), Nuffield Department of Clinical Neurosciences, Oxford Molecular Pathology Institute, Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.

Light plays a critical role in the regulation of numerous aspects of physiology and behaviour, including the entrainment of circadian rhythms and the regulation of sleep. These responses involve melanopsin (OPN4)-expressing photosensitive retinal ganglion cells (pRGCs) in addition to rods and cones. Nocturnal light exposure in rodents has been shown to result in rapid sleep induction, in which melanopsin plays a key role. However, studies have also shown that light exposure can result in elevated corticosterone, a response that is not compatible with sleep. To investigate these contradictory findings and to dissect the relative contribution of pRGCs and rods/cones, we assessed the effects of light of different wavelengths on behaviourally defined sleep. Here, we show that blue light (470 nm) causes behavioural arousal, elevating corticosterone and delaying sleep onset. By contrast, green light (530 nm) produces rapid sleep induction. Compared to wildtype mice, these responses are altered in melanopsin-deficient mice (Opn4-/-), resulting in enhanced sleep in response to blue light but delayed sleep induction in response to green or white light. We go on to show that blue light evokes higher Fos induction in the SCN compared to the sleep-promoting ventrolateral preoptic area (VLPO), whereas green light produced greater responses in the VLPO. Collectively, our data demonstrates that nocturnal light exposure can have either an arousal- or sleep-promoting effect, and that these responses are melanopsin-mediated via different neural pathways with different spectral sensitivities. These findings raise important questions relating to how artificial light may alter behaviour in both the work and domestic setting.
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http://dx.doi.org/10.1371/journal.pbio.1002482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4898879PMC
June 2016

Loss-of-Function Mutations in FRRS1L Lead to an Epileptic-Dyskinetic Encephalopathy.

Am J Hum Genet 2016 06 26;98(6):1249-1255. Epub 2016 May 26.

Mammalian Genetics Unit, Medical Research Council Harwell, Oxfordshire OX11 ORD, UK.

Glutamatergic neurotransmission governs excitatory signaling in the mammalian brain, and abnormalities of glutamate signaling have been shown to contribute to both epilepsy and hyperkinetic movement disorders. The etiology of many severe childhood movement disorders and epilepsies remains uncharacterized. We describe a neurological disorder with epilepsy and prominent choreoathetosis caused by biallelic pathogenic variants in FRRS1L, which encodes an AMPA receptor outer-core protein. Loss of FRRS1L function attenuates AMPA-mediated currents, implicating chronic abnormalities of glutamatergic neurotransmission in this monogenic neurological disease of childhood.
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http://dx.doi.org/10.1016/j.ajhg.2016.04.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4908178PMC
June 2016

Reciprocal interactions between circadian clocks and aging.

Mamm Genome 2016 08 2;27(7-8):332-40. Epub 2016 May 2.

Sleep and Circadian Neuroscience Institute (SCNi), Nuffield Department of Clinical Neurosciences, Oxford Molecular Pathology Institute, Sir William Dunn School of Pathology, South Parks Road, Oxfordshire, OX1 3RE, UK.

Virtually, all biological processes in the body are modulated by an internal circadian clock which optimizes physiological and behavioral performance according to the changing demands of the external 24-h world. This circadian clock undergoes a number of age-related changes, at both the physiological and molecular levels. While these changes have been considered to be part of the normal aging process, there is increasing evidence that disruptions to the circadian system can substantially impact upon aging and these impacts will have clear health implications. Here we review the current data of how both the physiological and core molecular clocks change with age and how feedback from external cues may modulate the aging of the circadian system.
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http://dx.doi.org/10.1007/s00335-016-9639-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4935744PMC
August 2016

Early doors (Edo) mutant mouse reveals the importance of period 2 (PER2) PAS domain structure for circadian pacemaking.

Proc Natl Acad Sci U S A 2016 Mar 22;113(10):2756-61. Epub 2016 Feb 22.

Medical Research Council Harwell, Harwell Science and Innovation Campus, Oxfordshire OX11 0RD, United Kingdom;

The suprachiasmatic nucleus (SCN) defines 24 h of time via a transcriptional/posttranslational feedback loop in which transactivation of Per (period) and Cry (cryptochrome) genes by BMAL1-CLOCK complexes is suppressed by PER-CRY complexes. The molecular/structural basis of how circadian protein complexes function is poorly understood. We describe a novel N-ethyl-N-nitrosourea (ENU)-induced mutation, early doors (Edo), in the PER-ARNT-SIM (PAS) domain dimerization region of period 2 (PER2) (I324N) that accelerates the circadian clock of Per2(Edo/Edo) mice by 1.5 h. Structural and biophysical analyses revealed that Edo alters the packing of the highly conserved interdomain linker of the PER2 PAS core such that, although PER2(Edo) complexes with clock proteins, its vulnerability to degradation mediated by casein kinase 1ε (CSNK1E) is increased. The functional relevance of this mutation is revealed by the ultrashort (<19 h) but robust circadian rhythms in Per2(Edo/Edo); Csnk1e(Tau/Tau) mice and the SCN. These periods are unprecedented in mice. Thus, Per2(Edo) reveals a direct causal link between the molecular structure of the PER2 PAS core and the pace of SCN circadian timekeeping.
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http://dx.doi.org/10.1073/pnas.1517549113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4791021PMC
March 2016

The Regulatory Factor ZFHX3 Modifies Circadian Function in SCN via an AT Motif-Driven Axis.

Cell 2015 Jul;162(3):607-21

MRC Harwell, Harwell Science and Innovation Campus, Oxfordshire OX11 0RD, UK. Electronic address:

We identified a dominant missense mutation in the SCN transcription factor Zfhx3, termed short circuit (Zfhx3(Sci)), which accelerates circadian locomotor rhythms in mice. ZFHX3 regulates transcription via direct interaction with predicted AT motifs in target genes. The mutant protein has a decreased ability to activate consensus AT motifs in vitro. Using RNA sequencing, we found minimal effects on core clock genes in Zfhx3(Sci/+) SCN, whereas the expression of neuropeptides critical for SCN intercellular signaling was significantly disturbed. Moreover, mutant ZFHX3 had a decreased ability to activate AT motifs in the promoters of these neuropeptide genes. Lentiviral transduction of SCN slices showed that the ZFHX3-mediated activation of AT motifs is circadian, with decreased amplitude and robustness of these oscillations in Zfhx3(Sci/+) SCN slices. In conclusion, by cloning Zfhx3(Sci), we have uncovered a circadian transcriptional axis that determines the period and robustness of behavioral and SCN molecular rhythms.
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http://dx.doi.org/10.1016/j.cell.2015.06.060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4537516PMC
July 2015

Analysis of mammalian gene function through broad-based phenotypic screens across a consortium of mouse clinics.

Nat Genet 2015 Sep 27;47(9):969-978. Epub 2015 Jul 27.

Institute for Medical Microbiology, Immunology and Hygiene, Technische Universität München, Munich, Germany.

The function of the majority of genes in the mouse and human genomes remains unknown. The mouse embryonic stem cell knockout resource provides a basis for the characterization of relationships between genes and phenotypes. The EUMODIC consortium developed and validated robust methodologies for the broad-based phenotyping of knockouts through a pipeline comprising 20 disease-oriented platforms. We developed new statistical methods for pipeline design and data analysis aimed at detecting reproducible phenotypes with high power. We acquired phenotype data from 449 mutant alleles, representing 320 unique genes, of which half had no previous functional annotation. We captured data from over 27,000 mice, finding that 83% of the mutant lines are phenodeviant, with 65% demonstrating pleiotropy. Surprisingly, we found significant differences in phenotype annotation according to zygosity. New phenotypes were uncovered for many genes with previously unknown function, providing a powerful basis for hypothesis generation and further investigation in diverse systems.
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http://dx.doi.org/10.1038/ng.3360DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4564951PMC
September 2015

A twin and molecular genetics study of sleep paralysis and associated factors.

J Sleep Res 2015 Aug 9;24(4):438-46. Epub 2015 Feb 9.

Department of Psychology, Goldsmiths, University of London, London, UK.

Sleep paralysis is a relatively common but under-researched phenomenon. In this paper we examine prevalence in a UK sample and associations with candidate risk factors. This is the first study to investigate the heritability of sleep paralysis in a twin sample and to explore genetic associations between sleep paralysis and a number of circadian expressed single nucleotide polymorphisms. Analyses are based on data from the Genesis1219 twin/sibling study, a community sample of twins/siblings from England and Wales. In total, data from 862 participants aged 22-32 years (34% male) were used in the study. This sample consisted of monozygotic and dizygotic twins and siblings. It was found that self-reports of general sleep quality, anxiety symptoms and exposure to threatening events were all associated independently with sleep paralysis. There was moderate genetic influence on sleep paralysis (53%). Polymorphisms in the PER2 gene were associated with sleep paralysis in additive and dominant models of inheritance-although significance was not reached once a Bonferroni correction was applied. It is concluded that factors associated with disrupted sleep cycles appear to be associated with sleep paralysis. In this sample of young adults, sleep paralysis was moderately heritable. Future work should examine specific polymorphisms associated with differences in circadian rhythms and sleep homeostasis further in association with sleep paralysis.
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http://dx.doi.org/10.1111/jsr.12282DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4950339PMC
August 2015

A novel SOD1-ALS mutation separates central and peripheral effects of mutant SOD1 toxicity.

Hum Mol Genet 2015 Apr 2;24(7):1883-97. Epub 2014 Dec 2.

MRC Mammalian Genetics Unit, Harwell, Oxfordshire OX11 0RD, UK,

Transgenic mouse models expressing mutant superoxide dismutase 1 (SOD1) have been critical in furthering our understanding of amyotrophic lateral sclerosis (ALS). However, such models generally overexpress the mutant protein, which may give rise to phenotypes not directly relevant to the disorder. Here, we have analysed a novel mouse model that has a point mutation in the endogenous mouse Sod1 gene; this mutation is identical to a pathological change in human familial ALS (fALS) which results in a D83G change in SOD1 protein. Homozgous Sod1(D83G/D83G) mice develop progressive degeneration of lower (LMN) and upper motor neurons, likely due to the same unknown toxic gain of function as occurs in human fALS cases, but intriguingly LMN cell death appears to stop in early adulthood and the mice do not become paralyzed. The D83 residue coordinates zinc binding, and the D83G mutation results in loss of dismutase activity and SOD1 protein instability. As a result, Sod1(D83G/D83G) mice also phenocopy the distal axonopathy and hepatocellular carcinoma found in Sod1 null mice (Sod1(-/-)). These unique mice allow us to further our understanding of ALS by separating the central motor neuron body degeneration and the peripheral effects from a fALS mutation expressed at endogenous levels.
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http://dx.doi.org/10.1093/hmg/ddu605DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4355022PMC
April 2015

Genetic background influences age-related decline in visual and nonvisual retinal responses, circadian rhythms, and sleep.

Neurobiol Aging 2015 Jan 2;36(1):380-93. Epub 2014 Aug 2.

MRC Harwell, Harwell Science and Innovation Campus, Oxfordshire, UK. Electronic address:

The circadian system is entrained to the environmental light/dark cycle via retinal photoreceptors and regulates numerous aspects of physiology and behavior, including sleep. These processes are all key factors in healthy aging showing a gradual decline with age. Despite their importance, the exact mechanisms underlying this decline are yet to be fully understood. One of the most effective tools we have to understand the genetic factors underlying these processes are genetically inbred mouse strains. The most commonly used reference mouse strain is C57BL/6J, but recently, resources such as the International Knockout Mouse Consortium have started producing large numbers of mouse mutant lines on a pure genetic background, C57BL/6N. Considering the substantial genetic diversity between mouse strains we expect there to be phenotypic differences, including differential effects of aging, in these and other strains. Such differences need to be characterized not only to establish how different mouse strains may model the aging process but also to understand how genetic background might modify age-related phenotypes. To ascertain the effects of aging on sleep/wake behavior, circadian rhythms, and light input and whether these effects are mouse strain-dependent, we have screened C57BL/6J, C57BL/6N, C3H-HeH, and C3H-Pde6b+ mouse strains at 5 ages throughout their life span. Our data show that sleep, circadian, and light input parameters are all disrupted by the aging process. Moreover, we have cataloged a number of strain-specific aging effects, including the rate of cataract development, decline in the pupillary light response, and changes in sleep fragmentation and the proportion of time spent asleep.
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http://dx.doi.org/10.1016/j.neurobiolaging.2014.07.040DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4270439PMC
January 2015

Polymorphisms in the circadian expressed genes PER3 and ARNTL2 are associated with diurnal preference and GNβ3 with sleep measures.

J Sleep Res 2014 Oct 17;23(5):595-604. Epub 2014 Mar 17.

MRC Harwell, Harwell Science and Innovation Campus, Oxfordshire, UK.

Sleep and circadian rhythms are intrinsically linked, with several sleep traits, including sleep timing and duration, influenced by both sleep homeostasis and the circadian phase. Genetic variation in several circadian genes has been associated with diurnal preference (preference in timing of sleep), although there has been limited research on whether they are associated with other sleep measurements. We investigated whether these genetic variations were associated with diurnal preference (Morningness-Eveningness Questionnaire) and various sleep measures, including: the global Pittsburgh Sleep Quality index score; sleep duration; and sleep latency and sleep quality. We genotyped 10 polymorphisms in genes with circadian expression in participants from the G1219 sample (n = 966), a British longitudinal population sample of young adults. We conducted linear regressions using dominant, additive and recessive models of inheritance to test for associations between these polymorphisms and the sleep measures. We found a significant association between diurnal preference and a polymorphism in period homologue 3 (PER3) (P < 0.005, recessive model) and a novel nominally significant association between diurnal preference and a polymorphism in aryl hydrocarbon receptor nuclear translocator-like 2 (ARNTL2) (P < 0.05, additive model). We found that a polymorphism in guanine nucleotide binding protein beta 3 (GNβ3) was associated significantly with global sleep quality (P < 0.005, recessive model), and that a rare polymorphism in period homologue 2 (PER2) was associated significantly with both sleep duration and quality (P < 0.0005, recessive model). These findings suggest that genes with circadian expression may play a role in regulating both the circadian clock and sleep homeostasis, and highlight the importance of further studies aimed at dissecting the specific roles that circadian genes play in these two interrelated but unique behaviours.
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http://dx.doi.org/10.1111/jsr.12144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4320759PMC
October 2014
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