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.

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.

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.

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.

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.

Uncovering the mechanisms that underlie central noradrenergic neuron heterogeneity is essential to understanding selective subtype vulnerability to disease and environmental insult. Using recombinase-based intersectional genetic fate mapping we have previously demonstrated that molecularly distinct progenitor populations give rise to mature noradrenergic neurons differing in their anatomical location, axon morphology and efferent projection pattern. Here we review the findings from our previous study and extend our analysis of the noradrenergic subpopulation defined by transient developmental expression of Hoxb1. Using a combination of intersectional genetic fate mapping and analysis of a targeted loss of function mutation in Hoxb1, we have now uncovered additional heterogeneity based on the requirement of some noradrenergic neurons for Hoxb1 expression. By comparing the distribution of noradrenergic neurons derived from the Hoxb1 expression domain in wild-type and mutant mice, we demonstrate that Hoxb1 expression is required by a subset of neurons in the pons. Additional fate mapping, using a Hoxb1 enhancer element that drives Cre recombinase expression exclusively in rhombomere 4 of the hindbrain, reveals the existence of a subpopulation of noradrenergic neurons in the pons with more restricted axonal targets than the full Hoxb1-derived subpopulation. The unique projection profile of this newly defined subpopulation suggests that it may be functionally distinct. These analyses shed new light on the molecular determinants of noradrenergic identity in the pons and the overall complexity of the central noradrenergic system. This article is part of a Special Issue entitled SI: Noradrenergic System.

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).

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.

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.

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.

This article describes an open-access gene expression database analyzed for more than 2,000 genes on mouse nervous system tissue in the coronal, sagittal, and transverse orientation representing multiple developmental ages.

Central norepinephrine-producing neurons comprise a diverse population of cells differing in anatomical location, connectivity, function and response to disease and environmental insult. The mechanisms that generate this diversity are unknown. Here we elucidate the lineal relationship between molecularly distinct progenitor populations in the developing mouse hindbrain and mature norepinephrine neuron subtype identity. We have identified four genetically separable subpopulations of mature norepinephrine neurons differing in their anatomical location, axon morphology and efferent projection pattern. One of the subpopulations showed an unexpected projection to the prefrontal cortex, challenging the long-held belief that the locus coeruleus is the sole source of norepinephrine projections to the cortex. These findings reveal the embryonic origins of central norepinephrine neurons and provide multiple molecular points of entry for future study of individual norepinephrine circuits in complex behavioral and physiological processes including arousal, attention, mood, memory, appetite and homeostasis.

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.

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.

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.

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.