A knock-in allele of Hand2 expressing Dre recombinase.

HAND2 is a basic helix-loop-helix transcription factor with diverse functions during development. To facilitate the investigation of genetic and functional diversity among Hand2-expressing cells in the mouse, we have generated Hand2Dre, a knock-in allele expressing Dre recombinase. To avoid disrupting Hand2 function, the Dre cDNA is inserted at the 3' end of the Hand2 coding sequence following a viral 2A peptide. Hand2Dre homozygotes can therefore be used in complex crosses to increase the proportion of useful genotypes among offspring. Dre expression in mid-gestation Hand2Dre embryos is indistinguishable from wild-type Hand2 expression, and HandDre efficiently recombines rox target sites in vivo. In combination with existing Cre and Flp mouse lines, Hand2Dre will therefore extend the ability to perform genetic intersectional labeling, fate mapping, and functional manipulation of subpopulations of cells characterized by developmental expression of Hand2.

Identifying diabetogenic drugs using real world health care databases: A Danish and Australian symmetry analysis.

AIMS: Drug-induced diabetes is underreported in conventional drug safety monitoring and may contribute to the increasing incidence of type 2 diabetes. Therefore, we used routinely collected prescription data to screen all commonly used drugs for diabetogenic effects. METHODS: Leveraging the Danish nationwide health registries, we used a case-only symmetry analysis design to evaluate all possible associations between drug initiation and subsequent diabetes. The study was conducted among individuals aged ≥40 years with a first-ever prescription for any antidiabetic drug 1996-2018 (n = 348 996). Sequence ratios (SRs) and 95% confidence intervals (CIs) were obtained for all possible drug class-diabetes combinations. A lower bound of the 95% CI >1.00 was considered a signal. Signals generated in Denmark were replicated using the Services Australia, Pharmaceutical Benefits Scheme 10% data extract. RESULTS: Overall, 386 drug classes were investigated, of which 70 generated a signal. In total, 43 were classified as previously known based on the SIDER database or a literature review, for example, glucocorticoids (SR 1.67, 95% CI 1.62-1.72) and ß-blockers (SR 1.20, 95% CI 1.16-1.23). Of 27 new signals, three drug classes yielded a signal in both the Danish and Australian data source: digitalis glycosides (SR 2.15, 95% CI 2.04-2.27, and SR 1.76, 95% CI 1.50-2.08), macrolides (SR 1.20, 95% CI 1.16-1.24, and SR 1.11, 95% CI 1.06-1.16) and inhaled ß2-agonists combined with glucocorticoids (SR 1.35, 95% CI 1.28-1.42, and SR 1.14, 95% CI 1.06-1.22). CONCLUSION: We identified 70 drug-diabetes associations, of which 27 were classified as hitherto unknown. Further studies evaluating the hypotheses generated by this work are needed, particularly for the signal for digitalis glycosides.

Assessment of Mapping the Brain, a Novel Research and Neurotechnology Based Approach for the Modern Neuroscience Classroom.

Neuroscience research is changing at an incredible pace due to technological innovation and recent national and global initiatives such as the BRAIN initiative. Given the wealth of data supporting the value of course-based undergraduate research experiences (CUREs) for students, we developed and assessed a neurotechnology CURE, Mapping the Brain. The goal of the course is to immerse undergraduate and graduate students in research and to explore technological advances in neuroscience. In the laboratory portion of the course, students pursued a hypothesis-driven, collaborative National Institutes of Health (NIH) research project. Using chemogenetic technology (Designer Receptors Exclusively Activated by Designer Drugs-DREADDs) and a recombinase-based intersectional genetic strategy, students mapped norepinephrine neurons, and their projections and explored the effects of activating these neurons in vivo. In lecture, students compared traditional and cutting-edge neuroscience methodologies, analyzed primary literature, designed hypothesis-based experiments, and discussed technological limitations of studying the brain. Over two consecutive years in the Program at North Carolina State University, we assessed student learning and perceptions of learning based on Society for Neuroscience's (SfN) core concepts and essential principles of neuroscience. Using analysis of student assignments and pre/post content and perception-based course surveys, we also assessed whether the course improved student research article analysis and neurotechnology assessment. Our analyses reveal new insights and pedagogical approaches for engaging students in research and improving their critical analysis of research articles and neurotechnologies. Our data also show that our multifaceted approach increased student confidence and promoted a data focused mentality when tackling research literature. Through the integration of authentic research and a neurotechnology focus, Mapping the Brain provides a unique model as a modern neuroscience laboratory course.

Redefining Noradrenergic Neuromodulation of Behavior: Impacts of a Modular Locus Coeruleus Architecture.

The locus coeruleus (LC) is a seemingly singular and compact neuromodulatory nucleus that is a prominent component of disparate theories of brain function due to its broad noradrenergic projections throughout the CNS. As a diffuse neuromodulatory system, noradrenaline affects learning and decision making, control of sleep and wakefulness, sensory salience including pain, and the physiology of correlated forebrain activity (ensembles and networks) and brain hemodynamic responses. However, our understanding of the LC is undergoing a dramatic shift due to the application of state-of-the-art methods that reveal a nucleus of many modules that provide targeted neuromodulation. Here, we review the evidence supporting a modular LC based on multiple levels of observation (developmental, genetic, molecular, anatomical, and neurophysiological). We suggest that the concept of the LC as a singular nucleus and, alongside it, the role of the LC in diverse theories of brain function must be reconsidered.

Genetic identification of a population of noradrenergic neurons implicated in attenuation of stress-related responses.

Noradrenergic signaling plays a well-established role in promoting the stress response. Here we identify a subpopulation of noradrenergic neurons, defined by developmental expression of Hoxb1, that has a unique role in modulating stress-related behavior. Using an intersectional chemogenetic strategy, in combination with behavioral and physiological analyses, we show that activation of Hoxb1-noradrenergic (Hoxb1-NE) neurons decreases anxiety-like behavior and promotes an active coping strategy in response to acute stressors. In addition, we use cerebral blood volume-weighted functional magnetic resonance imaging to show that chemoactivation of Hoxb1-NE neurons results in reduced activity in stress-related brain regions, including the bed nucleus of the stria terminalis, amygdala, and locus coeruleus. Thus, the actions of Hoxb1-NE neurons are distinct from the well-documented functions of the locus coeruleus in promoting the stress response, demonstrating that the noradrenergic system contains multiple functionally distinct subpopulations.

Use of vitamin K antagonists and risk of prostate cancer: Meta-analysis and nationwide case-control study.

Use of vitamin K antagonists (VKAs) has been suggested to reduce the risk of prostate cancer. We conducted a nested case-control study using Danish demographic and health data registries and summarized existing evidence in a meta-analysis. The case-control study included all Danish men aged 40-85 years with incident histologically verified prostate adenocarcinoma between 2005 and 2015 (cases). For each case, we selected 10 age-matched controls. We used conditional logistic regression to estimate odds ratios (ORs) with 95% confidence intervals (CI) for prostate cancer associated with long-term VKA use adjusted for concomitant drug use, medical history and socioeconomic status. Among 38,832 prostate cancer cases, 1,089 (2.8%) had used VKAs for 3 or more years compared to 10,803 (2.8%) controls yielding a crude OR of 1.01 (95% CI, 0.95-1.08). Multivariable adjustment for covariates had limited influence on the association (OR, 1.03; 95% CI, 0.97-1.10). We observed no dose-response relationship (e.g. OR for 5-10 years of use, 1.06 95% CI, 0.97-1.16). We included 8 studies in the meta-analysis reporting effect estimates from 0.51 (95% CI, 0.23-1.13) to 1.10 (95% CI, 0.94-1.40). Using random effect methods, a pooled effect estimate of 0.86 (95% CI, 0.70-1.05) was obtained; however, there was considerable across-study heterogeneity (I2 : 93.9%). In conclusion, we did not observe a reduced risk of prostate cancer associated with VKA use in this nationwide study and, taken together with previous study findings, a major protective effect of VKAs against prostate cancer seems unlikely.

Expanding the power of recombinase-based labeling to uncover cellular diversity.

Investigating the developmental, structural and functional complexity of mammalian tissues and organs depends on identifying and gaining experimental access to diverse cell populations. Here, we describe a set of recombinase-responsive fluorescent indicator alleles in mice that significantly extends our ability to uncover cellular diversity by exploiting the intrinsic genetic signatures that uniquely define cell types. Using a recombinase-based intersectional strategy, these new alleles uniquely permit non-invasive labeling of cells defined by the overlap of up to three distinct gene expression domains. In response to different combinations of Cre, Flp and Dre recombinases, they express eGFP and/or tdTomato to allow the visualization of full cellular morphology. Here, we demonstrate the value of these features through a proof-of-principle analysis of the central noradrenergic system. We label previously inaccessible subpopulations of noradrenergic neurons to reveal details of their three-dimensional architecture and axon projection profiles. These new indicator alleles will provide experimental access to cell populations at unprecedented resolution, facilitating analysis of their developmental origin and anatomical, molecular and physiological properties.

A new mouse line for cell ablation by diphtheria toxin subunit A controlled by a Cre-dependent FLEx switch.

Recombinase responsive mouse lines expressing diphtheria toxin subunit A (DTA) are well established tools for targeted ablation of genetically defined cell populations. Here we describe a new knock-in allele at the Gt(Rosa)26Sor locus that retains the best features of previously described DTA alleles-including a CAG promoter, attenuated mutant DTA cDNA, and ubiquitous EGFP labeling-with the addition of a Cre-dependent FLEx switch for tight control of expression. The FLEx switch consists of two pairs of antiparallel lox sites requiring Cre-mediated recombination for inversion of the DTA to the proper orientation for transcription. We demonstrate its utility by Cre-dependent ablation of both a broad domain in the embryonic nervous system and a discrete population of cells in the fetal gonads. We conclude that this new DTA line is useful for targeted ablation of genetically-defined cell populations.

A knock-in allele of En1 expressing dre recombinase.

Engrailed 1 (En1) is a homeobox-containing transcription factor expressed during development in diverse tissues, including the embryonic midbrain and anterior hindbrain. To facilitate investigation of genetic and developmental heterogeneity among cells with a history of En1 expression, we have generated En1(Dre) , a knock-in allele expressing Dre recombinase. En1(Dre) can be used with existing Cre and Flp recombinase lines for genetic intersectional labeling, fate mapping, and functional manipulation of subpopulations of cells characterized by transient expression of En1. To avoid disrupting En1 function, the Dre cDNA is inserted at the 3' end of the En1 coding sequence, together with a viral 2A peptide to mediate translation of separate EN1 and Dre proteins. Consequently, viable and fertile En1(Dre) homozygotes can be used to increase the proportion of useful genotypes produced in complex crosses. The pattern of Dre expression from En1(Dre) is indistinguishable from wild-type En1 expression in mid-gestation mouse embryos, and En1(Dre) controls Dre-responsive indicator alleles by efficiently recombining rox sites in vivo. Through the application of genetic tools that allow manipulation of cells based on combinatorial expression of multiple distinct recombinases, En1(Dre) will significantly extend the ability to target important subpopulations of neurons and other cells within the broader En1 expression domain. genesis 54:447-454, 2016. Published 2016. This article is a US Government work and is in the public domain in the USA.

Partial or Complete Loss of Norepinephrine Differentially Alters Contextual Fear and Catecholamine Release Dynamics in Hippocampal CA1.

Background: Contextual fear learning is heavily dependent on the hippocampus. Despite evidence that catecholamines contribute to contextual encoding and memory retrieval, the precise temporal dynamics of their release in the hippocampus during behavior is unknown. In addition, new animal models are required to probe the effects of altered catecholamine synthesis on release dynamics and contextual learning. Methods: We generated 2 new mouse models of altered locus coeruleus-norepinephrine (NE) synthesis and utilized them together with GRABNE and GRABDA sensors and in vivo fiber photometry to investigate NE and dopamine (DA) release dynamics in the dorsal hippocampal CA1 during contextual fear conditioning. Results: Aversive foot shock increased both NE and DA release in the dorsal CA1, while freezing behavior associated with recall of fear memory was accompanied by decreased release. Moreover, we found that freezing at the recent time point was sensitive to both partial and complete loss of locus coeruleus-NE synthesis throughout prenatal and postnatal development, similar to previous observations of mice with global loss of NE synthesis beginning postnatally. In contrast, freezing at the remote time point was compromised only by complete loss of locus coeruleus-NE synthesis beginning prenatally. Conclusions: Overall, these findings provide novel insights into the role of NE in contextual fear and the precise temporal dynamics of both NE and DA during freezing behavior and highlight complex relationships between genotype, sex, and NE signaling.

Locus coeruleus noradrenaline depletion and its differential impact on CO2-induced panic and hyperventilation in male and female mice.

CO2 exposure has been used to investigate the panicogenic response in patients with panic disorder. These patients are more sensitive to CO2, and more likely to experience the "false suffocation alarm" which triggers panic attacks. Imbalances in locus coeruleus noradrenergic (LC-NA) neurotransmission are responsible for psychiatric disorders, including panic disorder. These neurons are sensitive to changes in CO2/pH. Therefore, we investigated if LC-NA neurons are differentially activated after severe hypercapnia in mice. Further, we evaluated the participation of LC-NA neurons in ventilatory and panic-like escape responses induced by 20% CO2 in male and female wild type mice and two mouse models of altered LC-NA synthesis. Hypercapnia activates the LC-NA neurons, with males presenting a heightened level of activation. Mutant males lacking or with reduced LC-NA synthesis showed hypoventilation, while animals lacking LC noradrenaline present an increased metabolic rate compared to wild type in normocapnia. When exposed to CO2, males lacking LC noradrenaline showed a lower respiratory frequency compared to control animals. On the other hand, females lacking LC noradrenaline presented a higher tidal volume. Nevertheless, no change in ventilation was observed in either sex. CO2 evoked an active escape response. Mice lacking LC noradrenaline had a blunted jumping response and an increased freezing duration compared to the other groups. They also presented fewer racing episodes compared to wild type animals, but not different from mice with reduced LC noradrenaline. These findings suggest that LC-NA has an important role in ventilatory and panic-like escape responses elicited by CO2 exposure in mice.

Radial glia cells are candidate stem cells of ependymoma.

Tumors of the same histologic type often comprise clinically and molecularly distinct subgroups; however, the etiology of these subgroups is unknown. Here, we report that histologically identical, but genetically distinct, ependymomas exhibit patterns of gene expression that recapitulate those of radial glia cells in the corresponding region of the central nervous system. Cancer stem cells isolated from ependymomas displayed a radial glia phenotype and formed tumors when orthotopically transplanted in mice. These findings identify restricted populations of radial glia cells as candidate stem cells of the different subgroups of ependymoma, and they support a general hypothesis that subgroups of the same histologic tumor type are generated by different populations of progenitor cells in the tissues of origin.

Projections and interconnections of genetically defined serotonin neurons in mice.

Brain serotonin neurons are heterogeneous and can be distinguished by several anatomical and physiological characteristics. Toward resolving this heterogeneity into classes of functional relevance, subtypes of mature serotonin neurons were previously identified based on gene expression differences initiated during development in different rhombomeric (r) segments of the hindbrain. This redefinition of mature serotonin neuron subtypes based on the criteria of genetic lineage, along with the enabling genetic fate mapping tools, now allows various functional properties, such as axonal projections, to be allocated onto these identified subtypes. Furthermore, our approach uniquely enables interconnections between the different serotonin neuron subtypes to be determined; this is especially relevant because serotonin neuron activity is regulated by several feedback mechanisms. We used intersectional and subtractive genetic fate mapping tools to generate three independent lines of mice in which serotonin neurons arising in different rhombomeric segments, either r1, r2 or both r3 and r5, were uniquely distinguished from all other serotonin neurons by their expression of enhanced green fluorescent protein. Each of these subgroups of serotonergic neurons had a unique combination of forebrain projection targets. Typically more than one subgroup innervated an individual target area. Unique patterns of interconnections between the different groups of serotonin neurons were also observed and these pathways could subserve feedback regulatory circuits. Overall, the current findings suggest that activation of subsets of serotonin neurons could result in topographic serotonin release in the forebrain coupled with feedback inhibition of serotonin neurons with alternative projection targets.

Redefining the serotonergic system by genetic lineage.

Central serotonin-producing neurons are heterogeneous-differing in location, morphology, neurotoxin sensitivity and associated clinical disorders-but the underpinnings of this heterogeneity are largely unknown, as are the markers that distinguish physiological subtypes of serotonergic neurons. Here we redefined serotonergic subtypes on the basis of genetic programs that are differentially enacted in progenitor cells. We uncovered a molecular framework for the serotonergic system that, having genetic lineages as its basis, is likely to have physiological relevance and will permit access to genetically defined subtypes for manipulation.

Probing the structure and function of locus coeruleus projections to CNS motor centers.

The brainstem nucleus locus coeruleus (LC) sends projections to the forebrain, brainstem, cerebellum and spinal cord and is a source of the neurotransmitter norepinephrine (NE) in these areas. For more than 50 years, LC was considered to be homogeneous in structure and function such that NE would be released uniformly and act simultaneously on the cells and circuits that receive LC projections. However, recent studies have provided evidence that LC is modular in design, with segregated output channels and the potential for differential release and action of NE in its projection fields. These new findings have prompted a radical shift in our thinking about LC operations and demand revision of theoretical constructs regarding impact of the LC-NE system on behavioral outcomes in health and disease. Within this context, a major gap in our knowledge is the relationship between the LC-NE system and CNS motor control centers. While we know much about the organization of the LC-NE system with respect to sensory and cognitive circuitries and the impact of LC output on sensory guided behaviors and executive function, much less is known about the role of the LC-NE pathway in motor network operations and movement control. As a starting point for closing this gap in understanding, we propose using an intersectional recombinase-based viral-genetic strategy TrAC (Tracing Axon Collaterals) as well as established ex vivo electrophysiological assays to characterize efferent connectivity and physiological attributes of mouse LC-motor network projection neurons. The novel hypothesis to be tested is that LC cells with projections to CNS motor centers are scattered throughout the rostral-caudal extent of the nucleus but collectively display a common set of electrophysiological properties. Additionally, we expect to find these LC projection neurons maintain an organized network of axon collaterals capable of supporting selective, synchronous release of NE in motor circuitries for the purpose of coordinately regulating operations across networks that are responsible for balance and movement dynamics. Investigation of this hypothesis will advance our knowledge of the role of the LC-NE system in motor control and provide a basis for treating movement disorders resulting from disease, injury, or normal aging.

Chemogenetic stimulation of tonic locus coeruleus activity strengthens the default mode network.

The default mode network (DMN) of the brain is functionally associated with a wide range of behaviors. In this study, we used functional magnetic resonance imaging (fMRI), positron emission tomography (PET), and spectral fiber photometry to investigate the selective neuromodulatory effect of norepinephrine (NE)-releasing noradrenergic neurons in the locus coeruleus (LC) on the mouse DMN. Chemogenetic-induced tonic LC activity decreased cerebral blood volume (CBV) and glucose uptake and increased synchronous low-frequency fMRI activity within the frontal cortices of the DMN. Fiber photometry results corroborated these findings, showing that LC-NE activation induced NE release, enhanced calcium-weighted neuronal spiking, and reduced CBV in the anterior cingulate cortex. These data suggest that LC-NE alters conventional coupling between neuronal activity and CBV in the frontal DMN. We also demonstrated that chemogenetic activation of LC-NE neurons strengthened functional connectivity within the frontal DMN, and this effect was causally mediated by reduced modulatory inputs from retrosplenial and hippocampal regions to the association cortices of the DMN.

Natural locus coeruleus dynamics during feeding.

Recent data demonstrate that noradrenergic neurons of the locus coeruleus (LC-NE) are required for fear-induced suppression of feeding, but the role of endogenous LC-NE activity in natural, homeostatic feeding remains unclear. Here, we found that LC-NE activity was suppressed during food consumption, and the magnitude of this neural response was attenuated as mice consumed more pellets throughout the session, suggesting that LC responses to food are modulated by satiety state. Visual-evoked LC-NE activity was also attenuated in sated mice, suggesting that satiety state modulates LC-NE encoding of multiple behavioral states. We also found that food intake could be attenuated by brief or longer durations of LC-NE activation. Last, we found that activation of the LC to the lateral hypothalamus pathway suppresses feeding and enhances avoidance and anxiety-like responding. Our findings suggest that LC-NE neurons modulate feeding by integrating both external cues (e.g., anxiogenic environmental cues) and internal drives (e.g., satiety).

A brain-specific pgc1α fusion transcript affects gene expression and behavioural outcomes in mice.

PGC1α is a transcriptional coactivator in peripheral tissues, but its function in the brain remains poorly understood. Various brain-specific Pgc1α isoforms have been reported in mice and humans, including two fusion transcripts (FTs) with non-coding repetitive sequences, but their function is unknown. The FTs initiate at a simple sequence repeat locus ∼570 Kb upstream from the reference promoter; one also includes a portion of a short interspersed nuclear element (SINE). Using publicly available genomics data, here we show that the SINE FT is the predominant form of Pgc1α in neurons. Furthermore, mutation of the SINE in mice leads to altered behavioural phenotypes and significant up-regulation of genes in the female, but not male, cerebellum. Surprisingly, these genes are largely involved in neurotransmission, having poor association with the classical mitochondrial or antioxidant programs. These data expand our knowledge on the role of Pgc1α in neuronal physiology and suggest that different isoforms may have distinct functions. They also highlight the need for further studies before modulating levels of Pgc1α in the brain for therapeutic purposes.

Can an office practice telephonic response meet the needs of a pandemic?

INTRODUCTION: the H1N1 (subtype hemagglutinin 1 neuraminidase 1) influenza pandemic of 2009 was associated with a large increase in demand for primary care office visits. However, many patients with H1N1 symptoms or exposure could be assessed and treated with telephone protocols. METHODS: specific H1N1 influenza telephone protocols were developed by Mayo Clinic physicians using Centers for Disease Control recommendations. Using symptom calls to a primary care practice in the United States, we captured nurse telephone triage recommendations, telephone antiviral prescriptions, and what callers would have done without telephone advice. We retrospectively analyzed all symptom calls from July 2009 through January 2010. RESULTS: call volume was 5,596 calls monthly during the peak influenza months, which was 56% above the monthly average of 3,595 calls for the nonpeak months (p < 0.001). The calls during October 2009 were 111% over the nonpeak months (p < 0.001). In October 2009, telephone triage nurses gave 412 prescriptions for antivirals accounting for 5.4% of calls and 39% of all telephonic prescriptions for that month. In the peak H1N1 month of October, there were 1,522 callers who intended to stay home for their care. For the same month, triage nurses suggested 3,250 of the callers stay home. For an October 2009 appointment capacity of 35,126 visit slots, a potential 5% capacity was preserved. CONCLUSIONS: a telephone triage solution for the acute demands of influenza H1N1 demonstrated how patients had needs met telephonically while preserving medical access for others.

An Intersectional Viral-Genetic Method for Fluorescent Tracing of Axon Collaterals Reveals Details of Noradrenergic Locus Coeruleus Structure.

Understanding the function of broadly projecting neurons depends on comprehensive knowledge of the distribution and targets of their axon collaterals. While retrograde tracers and, more recently, retrograde viral vectors have been used to identify efferent projections, they have limited ability to reveal the full pattern of axon collaterals from complex, heterogeneous neuronal populations. Here we describe TrAC (tracing axon collaterals), an intersectional recombinase-based viral-genetic strategy that allows simultaneous visualization of axons from a genetically defined neuronal population and a projection-based subpopulation. To test this new method, we have applied TrAC to analysis of locus coeruleus norepinephrine (LC-NE)-containing neurons projecting to medial prefrontal cortex (mPFC) and primary motor cortex (M1) in laboratory mice. TrAC allowed us to label each projection-based LC-NE subpopulation, together with all remaining LC-NE neurons, in isolation from other noradrenergic populations. This analysis revealed mPFC-projecting and M1-projecting LC-NE subpopulations differ from each other and from the LC as a whole in their patterns of axon collateralization. Thus, TrAC complements and extends existing axon tracing methods by permitting analyses that have not previously been possible with complex genetically defined neuronal populations.