Now showing items 1-20 of 270

    • Shear stress activates nociceptors to drive Drosophila mechanical nociception

      Gong, Jiaxin; Chen, Jiazhang; Gu, Pengyu; Shang, Ye; Ruppell, Kendra Takle; Yang, Ying; Wang, Fei; Wen, Qi; Xiang, Yang (2022-09-02)
      Mechanical nociception is essential for animal survival. However, the forces involved in nociceptor activation and the underlying mechanotransduction mechanisms remain elusive. Here, we address these problems by investigating nocifensive behavior in Drosophila larvae. We show that strong poking stimulates nociceptors with a mixture of forces including shear stress and stretch. Unexpectedly, nociceptors are selectively activated by shear stress, but not stretch. Both the shear stress responses of nociceptors and nocifensive behavior require transient receptor potential A1 (TrpA1), which is specifically expressed in nociceptors. We further demonstrate that expression of mammalian or Drosophila TrpA1 in heterologous cells confers responses to shear stress but not stretch. Finally, shear stress activates TrpA1 in a membrane-delimited manner, through modulation of membrane fluidity. Together, our study reveals TrpA1 as an evolutionarily conserved mechanosensitive channel specifically activated by shear stress and suggests a critical role of shear stress in activating nociceptors to drive mechanical nociception.
    • Validation of DREADD agonists and administration route in a murine model of sleep enhancement

      Ferrari, Loris L; Ogbeide-Latario, Oghomwen E; Gompf, Heinrich S; Anaclet, Christelle (2022-07-30)
      Chemogenetics is a powerful tool to study the role of specific neuronal populations in physiology and diseases. Of particular interest, in mice, acute and specific activation of parafacial zone (PZ) GABAergic neurons expressing the Designer Receptors Activated by Designer Drugs (DREADD) hM3Dq (PZGABA-hM3Dq) enhances slow-wave-sleep (SWS), and this effect lasts for up to 6 h, allowing prolonged and detailed study of SWS. However, the most widely used DREADDs ligand, clozapine N-oxide (CNO), is metabolized into clozapine which has the potential of inducing non-specific effects. In addition, CNO is usually injected intraperitoneally (IP) in mice, limiting the number and frequency of repeated administration.
    • Elevated TNF-α Leads to Neural Circuit Instability in the Absence of Interferon Regulatory Factor 8

      Feinberg, Philip A; Becker, Shannon C; Chung, Leeyup; Ferrari, Loris; Stellwagen, David; Anaclet, Christelle; Durán-Laforet, Violeta; Faust, Travis E; Sumbria, Rachita K; Schafer, Dorothy P. (2022-07-05)
      Interferon regulatory factor 8 (IRF8) is a transcription factor necessary for the maturation of microglia, as well as other peripheral immune cells. It also regulates the transition of microglia and other immune cells to a pro-inflammatory phenotype. Irf8 is also a known risk gene for multiple sclerosis and lupus, and it has recently been shown to be downregulated in schizophrenia. While most studies have focused on IRF8-dependent regulation of immune cell function, little is known about how it impacts neural circuits. Here, we show by RNAseq from Irf8 -/- male and female mouse brains that several genes involved in regulation of neural activity are dysregulated. We then show that these molecular changes are reflected in heightened neural excitability and a profound increase in susceptibility to lethal seizures in male and female Irf8 -/- mice. Finally, we identify that TNF-α is elevated specifically in microglia in the CNS, and genetic or acute pharmacological blockade of TNF-α in the Irf8 -/- CNS rescued the seizure phenotype. These results provide important insights into the consequences of IRF8 signaling and TNF-α on neural circuits. Our data further suggest that neuronal function is impacted by loss of IRF8, a factor involved in neuropsychiatric and neurodegenerative diseases.SIGNIFICANCE STATEMENT Here, we identify a previously unknown and key role for interferon regulator factor 8 (IRF8) in regulating neural excitability and seizures. We further determine that these effects on neural circuits are through elevated TNF-α in the CNS. As IRF8 has most widely been studied in the context of regulating the development and inflammatory signaling in microglia and other immune cells, we have uncovered a novel function. Further, IRF8 is a risk gene for multiple sclerosis and lupus, IRF8 is dysregulated in schizophrenia, and elevated TNF-α has been identified in a multitude of neurologic conditions. Thus, elucidating these IRF8 and TNF-α-dependent effects on brain circuit function has profound implications for understanding underlying, therapeutically relevant mechanisms of disease.
    • Age-associated changes to neuronal dynamics involve a disruption of excitatory/inhibitory balance in C. elegans

      Wirak, Gregory S; Florman, Jeremy; Alkema, Mark J.; Connor, Christopher W; Gabel, Christopher V (2022-06-15)
      In the aging brain, many of the alterations underlying cognitive and behavioral decline remain opaque. Caenorhabditis elegans offers a powerful model for aging research, with a simple, well-studied nervous system to further our understanding of the cellular modifications and functional alterations accompanying senescence. We perform multi-neuronal functional imaging across the aged C. elegans nervous system, measuring an age-associated breakdown in system-wide functional organization. At single-cell resolution, we detect shifts in activity dynamics toward higher frequencies. In addition, we measure a specific loss of inhibitory signaling that occurs early in the aging process and alters the systems' critical excitatory/inhibitory balance. These effects are recapitulated with mutation of the calcium channel subunit UNC-2/CaV2α. We find that manipulation of inhibitory GABA signaling can partially ameliorate or accelerate the effects of aging. The effects of aging are also partially mitigated by disruption of the insulin signaling pathway, known to increase longevity, or by a reduction of caspase activation. Data from mammals are consistent with our findings, suggesting a conserved shift in the balance of excitatory/inhibitory signaling with age that leads to breakdown in global neuronal dynamics and functional decline.
    • PERIOD Phosphoclusters Control Temperature Compensation of the Circadian Clock

      Joshi, Radhika; Cai, Yao D; Xia, Yongliang; Chiu, Joanna C; Emery, Patrick (2022-06-02)
      Ambient temperature varies constantly. However, the period of circadian pacemakers is remarkably stable over a wide-range of ecologically- and physiologically-relevant temperatures, even though the kinetics of most biochemical reactions accelerates as temperature rises. This thermal buffering phenomenon, called temperature compensation, is a critical feature of circadian rhythms, but how it is achieved remains elusive. Here, we uncovered the important role played by the Drosophila PERIOD (PER) phosphodegron in temperature compensation. This phosphorylation hotspot is crucial for PER proteasomal degradation and is the functional homolog of mammalian PER2 S478 phosphodegron, which also impacts temperature compensation. Using CRISPR-Cas9, we introduced a series of mutations that altered three Serines of the PER phosphodegron. While all three Serine to Alanine substitutions lengthened period at all temperatures tested, temperature compensation was differentially affected. S44A and S45A substitutions caused undercompensation, while S47A resulted in overcompensation. These results thus reveal unexpected functional heterogeneity of phosphodegron residues in thermal compensation. Furthermore, mutations impairing phosphorylation of the per s phosphocluster showed undercompensation, consistent with its inhibitory role on S47 phosphorylation. We observed that S47A substitution caused increased accumulation of hyper-phosphorylated PER at warmer temperatures. This finding was corroborated by cell culture assays in which S47A slowed down phosphorylation-dependent PER degradation at high temperatures, causing PER degradation to be excessively temperature-compensated. Thus, our results point to a novel role of the PER phosphodegron in temperature compensation through temperature-dependent modulation of the abundance of hyper-phosphorylated PER. Our work reveals interesting mechanistic convergences and differences between mammalian and Drosophila temperature compensation of the circadian clock.
    • CPEB1 regulates the inflammatory immune response, phagocytosis, and alternative polyadenylation in microglia

      Ivshina, Maria P; van 't Spijker, Heleen M; Jung, Suna; Ponny, Sithara Raju; Schafer, Dorothy P.; Richter, Joel D (2022-05-30)
      Microglia are myeloid cells of the central nervous system that perform tasks essential for brain development, neural circuit homeostasis, and neural disease. Microglia react to inflammatory stimuli by upregulating inflammatory signaling through several different immune cell receptors such as the Toll-like receptor 4 (TLR4), which signals to several downstream effectors including transforming growth factor beta-activated kinase 1 (TAK1). Here, we show that TAK1 levels are regulated by CPEB1, a sequence-specific RNA binding protein that controls translation as well as RNA splicing and alternative poly(A) site selection in microglia. Lipopolysaccharide (LPS) binds the TLR4 receptor, which in CPEB1-deficient mice leads to elevated expression of ionized calcium binding adaptor molecule 1 (Iba1), a microglial protein that increases with inflammation, and increased levels of the cytokine IL6. This LPS-induced IL6 response is blocked by inhibitors of JNK, p38, ERK, NFκB, and TAK1. In contrast, phagocytosis, which is elevated in CPEB1-deficient microglia, is unaffected by LPS treatment or ERK inhibition, but is blocked by TAK1 inhibition. These data indicate that CPEB1 regulates microglial inflammatory responses and phagocytosis. RNA-seq indicates that these changes in inflammation and phagocytosis are accompanied by changes in RNA levels, splicing, and alternative poly(A) site selection. Thus, CPEB1 regulation of RNA expression plays a role in microglial function.
    • Astrocytic GABA transporter controls sleep by modulating GABAergic signaling in Drosophila circadian neurons

      Chaturvedi, Ratna; Stork, Tobias; Yuan, Chunyan; Freeman, Marc R.; Emery, Patrick (2022-03-17)
      A precise balance between sleep and wakefulness is essential to sustain a good quality of life and optimal brain function. GABA is known to play a key and conserved role in sleep control, and GABAergic tone should, therefore, be tightly controlled in sleep circuits. Here, we examined the role of the astrocytic GABA transporter (GAT) in sleep regulation using Drosophila melanogaster. We found that a hypomorphic gat mutation (gat(33-1)) increased sleep amount, decreased sleep latency, and increased sleep consolidation at night. Interestingly, sleep defects were suppressed when gat(33-1) was combined with a mutation disrupting wide-awake (wake), a gene that regulates the cell-surface levels of the GABAA receptor resistance to dieldrin (RDL) in the wake-promoting large ventral lateral neurons (l-LNvs). Moreover, RNAi knockdown of rdl and its modulators dnlg4 and wake in these circadian neurons also suppressed gat(33-1) sleep phenotypes. Brain immunohistochemistry showed that GAT-expressing astrocytes were located near RDL-positive l-LNv cell bodies and dendritic processes. We concluded that astrocytic GAT decreases GABAergic tone and RDL activation in arousal-promoting LNvs, thus determining proper sleep amount and quality in Drosophila.
    • Co-transmission of neuropeptides and monoamines choreograph the C. elegans escape response

      Florman, Jeremy T.; Alkema, Mark J. (2022-03-03)
      Co-localization and co-transmission of neurotransmitters and neuropeptides is a core property of neural signaling across species. While co-transmission can increase the flexibility of cellular communication, understanding the functional impact on neural dynamics and behavior remains a major challenge. Here we examine the role of neuropeptide/monoamine co-transmission in the orchestration of the C. elegans escape response. The tyraminergic RIM neurons, which coordinate distinct motor programs of the escape response, also co-express the neuropeptide encoding gene flp-18. We find that in response to a mechanical stimulus, flp-18 mutants have defects in locomotory arousal and head bending that facilitate the omega turn. We show that the induction of the escape response leads to the release of FLP-18 neuropeptides. FLP-18 modulates the escape response through the activation of the G-protein coupled receptor NPR-5. FLP-18 increases intracellular calcium levels in neck and body wall muscles to promote body bending. Our results show that FLP-18 and tyramine act in different tissues in both a complementary and antagonistic manner to control distinct motor programs during different phases of the C. elegans flight response. Our study reveals basic principles by which co-transmission of monoamines and neuropeptides orchestrate in arousal and behavior in response to stress.
    • Cell-Type-Specific Circadian Bioluminescence Rhythms in Dbp Reporter Mice

      Smith, Ciearra B.; van der Vinne, Vincent; McCartney, Eleanor; Stowie, Adam C.; Leise, Tanya L.; Martin-Burgos, Blanca; Molyneux, Penny C.; Garbutt, Lauren A.; Brodsky, Michael H.; Davidson, Alec J.; et al. (2022-02-01)
      Circadian rhythms are endogenously generated physiological and molecular rhythms with a cycle length of about 24 h. Bioluminescent reporters have been exceptionally useful for studying circadian rhythms in numerous species. Here, we report development of a reporter mouse generated by modification of a widely expressed and highly rhythmic gene encoding D-site albumin promoter binding protein (Dbp). In this line of mice, firefly luciferase is expressed from the Dbp locus in a Cre recombinase-dependent manner, allowing assessment of bioluminescence rhythms in specific cellular populations. A mouse line in which luciferase expression was Cre-independent was also generated. The Dbp reporter alleles do not alter Dbp gene expression rhythms in liver or circadian locomotor activity rhythms. In vivo and ex vivo studies show the utility of the reporter alleles for monitoring rhythmicity. Our studies reveal cell-type-specific characteristics of rhythms among neuronal populations within the suprachiasmatic nuclei ex vivo. In vivo studies show Dbp-driven bioluminescence rhythms in the liver of Albumin-Cre;DbpKI/+ "liver reporter" mice. After a shift of the lighting schedule, locomotor activity achieved the proper phase relationship with the new lighting cycle more rapidly than hepatic bioluminescence did. As previously shown, restricting food access to the daytime altered the phase of hepatic rhythmicity. Our model allowed assessment of the rate of recovery from misalignment once animals were provided with food ad libitum. These studies confirm the previously demonstrated circadian misalignment following environmental perturbations and reveal the utility of this model for minimally invasive, longitudinal monitoring of rhythmicity from specific mouse tissues.
    • A conserved neuropeptide system links head and body motor circuits to enable adaptive behavior

      Ramachandran, Shankar; Banerjee, Navonil; Bhattacharya, Raja; Lemons, Michele L.; Florman, Jeremy; Lambert, Christopher M.; Touroutine, Denis; Alexander, Kellianne; Schoofs, Liliane; Alkema, Mark J.; et al. (2021-11-12)
      Neuromodulators promote adaptive behaviors that are often complex and involve concerted activity changes across circuits that are often not physically connected. It is not well understood how neuromodulatory systems accomplish these tasks. Here, we show that the Caenorhabditis elegans NLP-12 neuropeptide system shapes responses to food availability by modulating the activity of head and body wall motor neurons through alternate G-protein coupled receptor (GPCR) targets, CKR-1 and CKR-2. We show ckr-2 deletion reduces body bend depth during movement under basal conditions. We demonstrate CKR-1 is a functional NLP-12 receptor and define its expression in the nervous system. In contrast to basal locomotion, biased CKR-1 GPCR stimulation of head motor neurons promotes turning during local searching. Deletion of ckr-1 reduces head neuron activity and diminishes turning while specific ckr-1 overexpression or head neuron activation promote turning. Thus, our studies suggest locomotor responses to changing food availability are regulated through conditional NLP-12 stimulation of head or body wall motor circuits.
    • Distinct neuropeptide-receptor modules regulate a sex-specific behavioral response to a pheromone

      Reilly, Douglas K.; McGlame, Emily J.; Vandewyer, Elke; Robidoux, Annalise N.; Muirhead, Caroline S.; Northcott, Haylea T.; Joyce, William; Alkema, Mark J.; Gegear, Robert J.; Beets, Isabel; et al. (2021-08-31)
      Dioecious species are a hallmark of the animal kingdom, with opposing sexes responding differently to identical sensory cues. Here, we study the response of C. elegans to the small-molecule pheromone, ascr#8, which elicits opposing behavioral valences in each sex. We identify a novel neuropeptide-neuropeptide receptor (NP/NPR) module that is active in males, but not in hermaphrodites. Using a novel paradigm of neuropeptide rescue that we established, we leverage bacterial expression of individual peptides to rescue the sex-specific response to ascr#8. Concurrent biochemical studies confirmed individual FLP-3 peptides differentially activate two divergent receptors, NPR-10 and FRPR-16. Interestingly, the two of the peptides that rescued behavior in our feeding paradigm are related through a conserved threonine, suggesting that a specific NP/NPR combination sets a male state, driving the correct behavioral valence of the ascr#8 response. Receptor expression within pre-motor neurons reveals novel coordination of male-specific and core locomotory circuitries.
    • The transcription factor LAG-1/CSL plays a Notch-independent role in controlling terminal differentiation, fate maintenance, and plasticity of serotonergic chemosensory neurons

      Maicas, Miren; Jimeno-Martin, Angela; Millan-Trejo, Andrea; Alkema, Mark J.; Flames, Nuria (2021-07-07)
      During development, signal-regulated transcription factors (TFs) act as basal repressors and upon signalling through morphogens or cell-to-cell signalling shift to activators, mediating precise and transient responses. Conversely, at the final steps of neuron specification, terminal selector TFs directly initiate and maintain neuron-type specific gene expression through enduring functions as activators. C. elegans contains 3 types of serotonin synthesising neurons that share the expression of the serotonin biosynthesis pathway genes but not of other effector genes. Here, we find an unconventional role for LAG-1, the signal-regulated TF mediator of the Notch pathway, as terminal selector for the ADF serotonergic chemosensory neuron, but not for other serotonergic neuron types. Regulatory regions of ADF effector genes contain functional LAG-1 binding sites that mediate activation but not basal repression. lag-1 mutants show broad defects in ADF effector genes activation, and LAG-1 is required to maintain ADF cell fate and functions throughout life. Unexpectedly, contrary to reported basal repression state for LAG-1 prior to Notch receptor activation, gene expression activation in the ADF neuron by LAG-1 does not require Notch signalling, demonstrating a default activator state for LAG-1 independent of Notch. We hypothesise that the enduring activity of terminal selectors on target genes required uncoupling LAG-1 activating role from receiving the transient Notch signalling.
    • A phase transition enhances the catalytic activity of SARM1, an NAD(+) glycohydrolase involved in neurodegeneration

      Loring, Heather S.; Czech, Victoria L.; Icso, Janneke D.; O'Connor, Lauren C.; Parelkar, Sangram; Byrne, Alexandra B.; Thompson, Paul R. (2021-06-29)
      Sterile alpha and toll/interleukin receptor (TIR) motif-containing protein 1 (SARM1) is a neuronally expressed NAD(+) glycohydrolase whose activity is increased in response to stress. NAD(+) depletion triggers axonal degeneration, which is a characteristic feature of neurological diseases. Notably, loss of SARM1 is protective in murine models of peripheral neuropathy and traumatic brain injury. Herein, we report that citrate induces a phase transition that enhances SARM1 activity by ~2000-fold. This phase transition can be disrupted by mutating a residue involved in multimerization, G601P. This mutation also disrupts puncta formation in cells. We further show that citrate induces axonal degeneration in C. elegans that is dependent on the C. elegans orthologue of SARM1 (TIR-1). Notably, citrate induces the formation of larger puncta indicating that TIR-1/SARM1 multimerization is essential for degeneration in vivo. These findings provide critical insights into SARM1 biology with important implications for the discovery of novel SARM1-targeted therapeutics.
    • Neuronal post-developmentally acting SAX-7S/L1CAM can function as cleaved fragments to maintain neuronal architecture in C. elegans

      Desse, Virginie E.; Blanchette, Cassandra R.; Nadour, Malika; Perrat, Paola N.; Rivollet, Lise; Khandekar, Anagha; Benard, Claire Y. (2021-06-11)
      Whereas remarkable advances have uncovered mechanisms that drive nervous system assembly, the processes responsible for the lifelong maintenance of nervous system architecture remain poorly understood. Subsequent to its establishment during embryogenesis, neuronal architecture is maintained throughout life in the face of the animal's growth, maturation processes, the addition of new neurons, body movements, and aging. The C. elegans protein SAX-7, homologous to the vertebrate L1 protein family of neural adhesion molecules, is required for maintaining the organization of neuronal ganglia and fascicles after their successful initial embryonic development. To dissect the function of sax-7 in neuronal maintenance, we generated a null allele and sax-7S-isoform-specific alleles. We find that the null sax-7(qv30) is, in some contexts, more severe than previously described mutant alleles, and that the loss of sax-7S largely phenocopies the null, consistent with sax-7S being the key isoform in neuronal maintenance. Using a sfGFP::SAX-7S knock-in, we observe sax-7S to be predominantly expressed across the nervous system, from embryogenesis to adulthood. Yet, its role in maintaining neuronal organization is ensured by post-developmentally acting SAX-7S, as larval transgenic sax-7S(+) expression alone is sufficient to profoundly rescue the null mutants' neuronal maintenance defects. Moreover, the majority of the protein SAX-7 appears to be cleaved, and we show that these cleaved SAX-7S fragments together, not individually, can fully support neuronal maintenance. These findings contribute to our understanding of the role of the conserved protein SAX-7/L1CAM in long-term neuronal maintenance, and may help decipher processes that go awry in some neurodegenerative conditions.
    • Corollary discharge promotes a sustained motor state in a neural circuit for navigation

      Ji, Ni; Venkatachalam, Vivek; Rodgers, Hillary Denise; Hung, Wesley; Kawano, Taizo; Clark, Christopher M.; Lim, Maria; Alkema, Mark J.; Zhen, Mei; Samuel, Aravinthan D. T. (2021-04-21)
      Animals exhibit behavioral and neural responses that persist on longer timescales than transient or fluctuating stimulus inputs. Here, we report that Caenorhabditis elegans uses feedback from the motor circuit to a sensory processing interneuron to sustain its motor state during thermotactic navigation. By imaging circuit activity in behaving animals, we show that a principal postsynaptic partner of the AFD thermosensory neuron, the AIY interneuron, encodes both temperature and motor state information. By optogenetic and genetic manipulation of this circuit, we demonstrate that the motor state representation in AIY is a corollary discharge signal. RIM, an interneuron that is connected with premotor interneurons, is required for this corollary discharge. Ablation of RIM eliminates the motor representation in AIY, allows thermosensory representations to reach downstream premotor interneurons, and reduces the animal's ability to sustain forward movements during thermotaxis. We propose that feedback from the motor circuit to the sensory processing circuit underlies a positive feedback mechanism to generate persistent neural activity and sustained behavioral patterns in a sensorimotor transformation.
    • Flexible motor sequence generation during stereotyped escape responses

      Wang, Yuan; Zhang, Xiaoqian; Xin, Qi; Hung, Wesley; Florman, Jeremy; Huo, Jing; Xu, Tianqi; Xie, Yu; Alkema, Mark J.; Zhen, Mei; et al. (2020-06-05)
      Complex animal behaviors arise from a flexible combination of stereotyped motor primitives. Here we use the escape responses of the nematode Caenorhabditis elegans to study how a nervous system dynamically explores the action space. The initiation of the escape responses is predictable: the animal moves away from a potential threat, a mechanical or thermal stimulus. But the motor sequence and the timing that follow are variable. We report that a feedforward excitation between neurons encoding distinct motor states underlies robust motor sequence generation, while mutual inhibition between these neurons controls the flexibility of timing in a motor sequence. Electrical synapses contribute to feedforward coupling whereas glutamatergic synapses contribute to inhibition. We conclude that C. elegans generates robust and flexible motor sequences by combining an excitatory coupling and a winner-take-all operation via mutual inhibition between motor modules.
    • A highly efficient method for single-cell electroporation in mouse organotypic hippocampal slice culture

      Keener, David G.; Cheung, Amy; Futai, Kensuke (2020-05-01)
      BACKGROUND: Exogenous gene introduction by transfection is one of the most important approaches for understanding the function of specific genes at the cellular level. Electroporation has a long-standing history as a versatile gene delivery technique in vitro and in vivo. However, it has been underutilized in vitro because of technical difficulty and insufficient transfection efficiency. NEW METHOD: We have developed an electroporation technique that combines the use of large glass electrodes, tetrodotoxin-containing artificial cerebrospinal fluid and mild electrical pulses. Here, we describe the technique and compare it with existing methods. RESULTS: Our method achieves a high transfection efficiency ( approximately 80 %) in both excitatory and inhibitory neurons with no detectable side effects on their function. We demonstrate this method is capable of transferring at least three different genes into a single neuron. In addition, we demonstrate the ability to transfect different genes into neighboring cells. COMPARISON WITH EXISTING METHODS: The majority of existing methods use fine-tipped glass electrodes (i.e. > 10MOmega) and apply high voltage (10V) pulses with high frequency (100Hz) for 1s. These parameters contribute to practical difficulties thus lowering the transfection efficiency. Our unique method minimizes electrode clogging and therefore procedure duration, increasing transfection efficiency and cellular viability. CONCLUSIONS: Our modifications, relative to current methods, optimize electroporation efficiency and cell survival. Our approach offers distinct research strategies not only in elucidating cell-autonomous functions of genes but also for assessing genes contributing to intercellular functions, such as trans-synaptic interactions.
    • Deconstructing circadian disruption: Assessing the contribution of reduced peripheral oscillator amplitude on obesity and glucose intolerance in mice

      van der Vinne, Vincent; Martin Burgos, Blanca; Harrington, Mary E.; Weaver, David R. (2020-04-03)
      Disturbing the circadian regulation of physiology by disruption of the rhythmic environment is associated with adverse health outcomes but the underlying mechanisms are unknown. Here, the response of central and peripheral circadian clocks to an advance or delay of the light-dark cycle was determined in mice. This identified transient damping of peripheral clocks as a consequence of an advanced light-dark cycle. Similar depression of peripheral rhythm amplitude was observed in mice exposed to repeated phase shifts. To assess the metabolic consequences of such peripheral amplitude depression in isolation, temporally chimeric mice lacking a functional central clock (Vgat-Cre(+) Bmal1(fl/fl) ) were housed in the absence of environmental rhythmicity. In vivo PER2::LUC bioluminescence imaging of anesthetized and freely moving mice revealed that this resulted in a state of peripheral amplitude depression, similar in severity to that observed transiently following an advance of the light-dark cycle. Surprisingly, our mice did not show alterations in body mass or glucose tolerance in males or females on regular or high-fat diets. Overall, our results identify transient damping of peripheral rhythm amplitude as a consequence of exposure to an advanced light-dark cycle but chronic damping of peripheral clocks in isolation is insufficient to induce adverse metabolic outcomes in mice.
    • In Situ Regulated Dopamine Transporter Trafficking: There's No Place Like Home

      Fagan, Rita R.; Kearney, Patrick J.; Melikian, Haley E. (2020-03-07)
      Dopamine (DA) is critical for motivation, reward, movement initiation, and learning. Mechanisms that control DA signaling have a profound impact on these important behaviors, and additionally play a role in DA-related neuropathologies. The presynaptic SLC6 DA transporter (DAT) limits extracellular DA levels by clearing released DA, and is potently inhibited by addictive and therapeutic psychostimulants. Decades of evidence support that the DAT is subject to acute regulation by a number of signaling pathways, and that endocytic trafficking strongly regulates DAT availability and function. DAT trafficking studies have been performed in a variety of model systems, including both in vitro and ex vivo preparations. In this review, we focus on the breadth of DAT trafficking studies, with specific attention to, and comparison of, how context may influence DAT's response to different stimuli. In particular, this overview highlights that stimulated DAT trafficking not only differs between in vitro and ex vivo environments, but also is influenced by both sex and anatomical subregions.
    • HIF-1 Has a Central Role in Caenorhabditis elegans Organismal Response to Selenium

      Romanelli-Credrez, Laura; Doitsidou, Maria; Alkema, Mark J.; Salinas, Gustavo (2020-02-25)
      Selenium is a trace element for most organisms; its deficiency and excess are detrimental. Selenium beneficial effects are mainly due to the role of the 21(st) genetically encoded amino acid selenocysteine (Sec). Selenium also exerts Sec-independent beneficial effects. Its harmful effects are thought to be mainly due to non-specific incorporation in protein synthesis. Yet the selenium response in animals is poorly understood. In Caenorhabditis elegans, Sec is genetically incorporated into a single selenoprotein. Similar to mammals, a 20-fold excess of the optimal selenium requirement is harmful. Sodium selenite (Na2SeO3) excess causes development retardation, impaired growth, and neurodegeneration of motor neurons. To study the organismal response to selenium we performed a genetic screen for C. elegans mutants that are resistant to selenite. We isolated non-sense and missense egl-9/EGLN mutants that confer robust resistance to selenium. In contrast, hif-1/HIF null mutant was highly sensitive to selenium, establishing a role for this transcription factor in the selenium response. We showed that EGL-9 regulates HIF-1 activity through VHL-1, and identified CYSL-1 as a key sensor that transduces the selenium signal. Finally, we showed that the key enzymes involved in sulfide and sulfite stress (sulfide quinone oxidoreductase and sulfite oxidase) are not required for selenium resistance. In contrast, knockout strains in the persulfide dioxygenase ETHE-1 and the sulfurtransferase MPST-7 affect the organismal response to selenium. In sum, our results identified a transcriptional pathway as well as enzymes possibly involved in the organismal selenium response.