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.

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.

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.

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.

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).

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.

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.

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.

Hippocampal Interneuronal α7 nAChRs Modulate Theta Oscillations in Freely Moving Mice.

Muscarinic acetylcholine receptors (mAChRs) are critically involved in hippocampal theta generation, but much less is known about the role of nicotinic AChRs (nAChRs). Here we provide evidence that α7 nAChRs expressed on interneurons, particularly those in oriens lacunosum moleculare (OLM), also regulate hippocampal theta generation. Local hippocampal infusion of a selective α7 nAChR antagonist significantly reduces hippocampal theta power and impairs Y-maze spontaneous alternation performance in freely moving mice. By knocking out receptors in different neuronal subpopulations, we find that α7 nAChRs expressed in OLM interneurons regulate theta generation. Our in vitro slice studies indicate that α7 nAChR activation increases OLM neuron activity that, in turn, enhances pyramidal cell excitatory postsynaptic currents (EPSCs). Our study also suggests that mAChR activation promotes transient theta generation, while α7 nAChR activation facilitates future theta generation by similar stimulations, revealing a complex mechanism whereby cholinergic signaling modulates different aspects of hippocampal theta oscillations through different receptor subtypes.

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.

Elimination of galanin synthesis in noradrenergic neurons reduces galanin in select brain areas and promotes active coping behaviors.

Accumulating evidence indicates that disruption of galanin signaling is associated with neuropsychiatric disease, but the precise functions of this neuropeptide remain largely unresolved due to lack of tools for experimentally disrupting its transmission in a cell type-specific manner. To examine the function of galanin in the noradrenergic system, we generated and crossed two novel knock-in mouse lines to create animals lacking galanin specifically in noradrenergic neurons (GalcKO-Dbh). We observed reduced levels of galanin peptide in pons, hippocampus, and prefrontal cortex of GalcKO-Dbh mice, indicating that noradrenergic neurons are a significant source of galanin to those brain regions, while midbrain and hypothalamic galanin levels were comparable to littermate controls. In these same brain regions, we observed no change in levels of norepinephrine or its major metabolite at baseline or after an acute stressor, suggesting that loss of galanin does not affect noradrenergic synthesis or turnover. GalcKO-Dbh mice had normal performance in tests of depression, learning, and motor-related behavior, but had an altered response in some anxiety-related tasks. Specifically, GalcKO-Dbh mice showed increased marble and shock probe burying and had a reduced latency to eat in a novel environment, indicative of a more proactive coping strategy. Together, these findings indicate that noradrenergic neurons provide a significant source of galanin to discrete brain areas, and noradrenergic-specific galanin opposes adaptive coping responses.

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.

Selective Activation of Cholecystokinin-Expressing GABA (CCK-GABA) Neurons Enhances Memory and Cognition.

Cholecystokinin-expressing GABAergic (CCK-GABA) neurons are perisomatic inhibitory cells that have been argued to regulate emotion and sculpt the network oscillations associated with cognition. However, no study has selectively manipulated CCK-GABA neuron activity during behavior in freely-moving animals. To explore the behavioral effects of activating CCK-GABA neurons on emotion and cognition, we utilized a novel intersectional genetic mouse model coupled with a chemogenetic approach. Specifically, we generated triple transgenic CCK-Cre;Dlx5/6-Flpe;RC::FL-hM3Dq (CCK-GABA/hM3Dq) mice that expressed the synthetic excitatory hM3Dq receptor in CCK-GABA neurons. Results showed that clozapine-N-oxide (CNO)-mediated activation of CCK-GABA neurons did not alter open field (OF) or tail suspension (TS) performance and only slightly increased anxiety in the elevated plus maze (EPM). Although CNO treatment had only modestly affected emotional behavior, it significantly enhanced multiple cognitive and memory behaviors including social recognition, contextual fear conditioning, contextual discrimination, object recognition, and problem-solving in the puzzle box. Collectively, these findings suggest that systemic activation of CCK-GABA neurons minimally affects emotion but significantly enhances cognition and memory. Our results imply that CCK-GABA neurons are more functionally diverse than originally expected and could serve as a potential therapeutic target for the treatment of cognitive/memory disorders.

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.

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.

CA2 neuronal activity controls hippocampal low gamma and ripple oscillations.

Hippocampal oscillations arise from coordinated activity among distinct populations of neurons and are associated with cognitive functions. Much progress has been made toward identifying the contribution of specific neuronal populations in hippocampal oscillations, but less is known about the role of hippocampal area CA2, which is thought to support social memory. Furthermore, the little evidence on the role of CA2 in oscillations has yielded conflicting conclusions. Therefore, we sought to identify the contribution of CA2 to oscillations using a controlled experimental system. We used excitatory and inhibitory DREADDs to manipulate CA2 neuronal activity and studied resulting hippocampal-prefrontal cortical network oscillations. We found that modification of CA2 activity bidirectionally regulated hippocampal and prefrontal cortical low-gamma oscillations and inversely modulated hippocampal ripple oscillations in mice. These findings support a role for CA2 in low-gamma generation and ripple modulation within the hippocampus and underscore the importance of CA2 in extrahippocampal oscillations.

DEFiNE: A Method for Enhancement and Quantification of Fluorescently Labeled Axons.

Visualization and quantification of fluorescently labeled axonal fibers are widely employed in studies of neuronal connectivity in the brain. However, accurate analysis of axon density is often confounded by autofluorescence and other fluorescent artifacts. By the time these problems are detected in labeled tissue sections, significant time and resources have been invested, and the tissue may not be easy to replace. In response to these difficulties, we have developed Digital Enhancement of Fibers with Noise Elimination (DEFiNE), a method for eliminating fluorescent artifacts from digital images based on their morphology and fluorescence spectrum, thus permitting enhanced visualization and quantification of axonal fibers. Application of this method is facilitated by a DEFiNE macro, written using ImageJ Macro Language (IJM), which includes an automated and customizable procedure for image processing and a semi-automated quantification method that accounts for any remaining local variation in background intensity. The DEFiNE macro is open-source and used with the widely available FIJI software for maximum accessibility.

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.

Two Subpopulations of Noradrenergic Neurons in the Locus Coeruleus Complex Distinguished by Expression of the Dorsal Neural Tube Marker Pax7.

Central noradrenergic neurons, collectively defined by synthesis of the neurotransmitter norepinephrine, are a diverse collection of cells in the hindbrain, differing in their anatomy, physiological and behavioral functions, and susceptibility to disease and environmental insult. To investigate the developmental basis of this heterogeneity, we have used an intersectional genetic fate mapping strategy in mice to study the dorsoventral origins of the En1-derived locus coeruleus (LC) complex which encompasses virtually all of the anatomically defined LC proper, as well as a portion of the A7 and subcoeruleus (SubC) noradrenergic nuclei. We show that the noradrenergic neurons of the LC complex originate in two different territories of the En1 expression domain in the embryonic hindbrain. Consistent with prior studies, we confirm that the majority of the LC proper arises from the alar plate, the dorsal domain of the neural tube, as defined by expression of Pax7Cre . In addition, our analysis shows that a large proportion of the En1-derived A7 and SubC nuclei also originate in the Pax7Cre -defined alar plate. Surprisingly, however, we identify a smaller subpopulation of the LC complex that arises from outside the Pax7Cre expression domain. We characterize the distribution of these neurons within the LC complex, their cell morphology, and their axonal projection pattern. Compared to the broader LC complex, the newly identified Pax7Cre -negative noradrenergic subpopulation has very sparse projections to thalamic nuclei, suggestive of distinct functions. This developmental genetic analysis opens new avenues of investigation into the functional diversity of the LC complex.