Scholarly Works, School of Neuroscience

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Kdo2-Lipid A of Escherichia coli, a defined endotoxin that activates macrophages via TLR-4
Raetz, CRH (2006-02-11)
TLR-4 and Sustained Calcium Agonists Synergistically Produce Eicosanoids Independent of Protein Synthesis in RAW264.7 Cells
Buczynski, Matthew W.; Stephens, Daren L.; Bowers-Gentry, Rebecca C.; Grkovich, Andrej; Deems, Raymond A.; Dennis, Edward A. (2007-08-03)
Arachidonic acid is released by phospholipaseA2 and converted into hundreds of distinct bioactive mediators by a variety of cyclooxygenases (COX), lipoxygenases (LO), and cytochrome P450s. Because of the size and diversity of the eicosanoid class of signaling molecules produced, a thorough and systematic investigation of these biological processes requires the simultaneous quantitation of a large number of eicosanoids in a single analysis. We have developed a robust liquid chromatography/tandem mass spectrometry method that can identify and quantitate over 60 different eicosanoids in a single analysis, and we applied it to agonist stimulated RAW264.7 murine macrophages. Fifteen different eicosanoids produced through COX and 5-LO were detected either intracellularly or in the media following stimulation with 16 different agonists of Toll-like receptors (TLR), G protein-coupled receptors, and purinergic receptors. No significant differences in the COX metabolite profiles were detected using the different agonists; however, we determined that only agonists creating a sustained Ca2²⁺ influx were capable of activating the 5-LO pathway in these cells. Synergy between Ca2²⁺ and TLR pathways was detected and discovered to be independent of NF-κB-induced protein synthesis. This demonstrates that TLR induction of protein synthesis and priming for enhanced phospholipase A₂-mediated eicosanoid production work through two distinct pathways.
Thematic Review Series: Proteomics. An integrated omics analysis of eicosanoid biology
Buczynski, Matthew W.; Dumlao, Darren S.; Dennis, Edward A. (2009-06-01)
Eicosanoids have been implicated in a vast number of devastating inflammatory conditions, including arthritis, atherosclerosis, pain, and cancer. Currently, over a hundred different eicosanoids have been identified, with many having potent bioactive signaling capacity. These lipid metabolites are synthesized de novo by at least 50 unique enzymes, many of which have been cloned and characterized. Due to the extensive characterization of eicosanoid biosynthetic pathways, this field provides a unique framework for integrating genomics, proteomics, and metabolomics toward the investigation of disease pathology. To facilitate a concerted systems biology approach, this review outlines the proteins implicated in eicosanoid biosynthesis and signaling in human, mouse, and rat. Applications of the extensive genomic and lipidomic research to date illustrate the questions in eicosanoid signaling that could be uniquely addressed by a thorough analysis of the entire eicosanoid proteome.
A developmental sex difference in hippocampal neurogenesis is mediated by endogenous Oestradiol
Bowers, J. Michael; Waddell, Jaylyn; McCarthy, Margaret M. (BMC, 2010)
Background: Oestradiol is a steroid hormone that exerts extensive influence on brain development and is a powerful modulator of hippocampal structure and function. The hippocampus is a critical brain region regulating complex cognitive and emotional responses and is implicated in the aetiology of several mental health disorders, many of which exhibit some degree of sex difference. Many sex differences in the adult rat brain are determined by oestradiol action during a sensitive period of development. We had previously reported a sex difference in rates of cell genesis in the developing hippocampus of the laboratory rat. Males generate more new cells on average than females. The current study explored the effects of both exogenous and endogenous oestradiol on this sex difference. Methods: New born male and female rat pups were injected with the mitotic marker 5-bromo-2-deoxyuridine (BrdU) and oestradiol or agents that antagonize oestradiol action. The effects on cell number, proliferation, differentiation and survival were assessed at several time points. Significant differences between groups were determined by two- or thee-Way ANOVA. Results: Newborn males had higher rates of cell proliferation than females. Oestradiol treatment increased cell proliferation in neonatal females, but not males, and in the CA1 region many of these cells differentiated into neurons. The increased rate of proliferation induced by neonatal oestradiol persisted until at least 3 weeks of age, suggesting an organizational effect. Administering the aromatase inhibitor, formestane, or the oestrogen receptor antagonist, tamoxifen, significantly decreased the number of new cells in males but not females. Conclusion: Endogenous oestradiol increased the rate of cell proliferation observed in newborn males compared to females. This sex difference in neonatal neurogenesis may have implications for adult differences in learning strategy, stress responsivity or vulnerability to damage or disease.
The role of the FOXP family of transcription factors in ASD
Bowers, J. Michael; Konopka, Genevieve (Hindawi, 2012)
Autism spectrum disorders (ASD) is a neurodevelopmental disease with complex genetics; however, the genes that are responsible for this disease still remain mostly unknown. Here, we focus on the FOXP family of transcription factors as there is emerging evidence strongly linking these genes to ASD and other genes implicated in ASD. The FOXP family of genes includes three genes expressed in the central nervous system: FOXP1, FOPX2, and FOXP4. This unique group of transcription factors has known functions in brain development as well as the evolution of language. We will also discuss the other genes including transcriptional targets of FOXP genes that have been found to be associated with language and may be important in the pathophysiology of ASD. Finally, we will review the emerging animal models currently being used to study the function of the FOXP genes within the context of ASD symptomology. The combination of gene expression and animal behavior is critical for elucidating how genes such as the FOXP family members are key players within the framework of the developing brain.
Sex, stress, and epigenetics: regulation of behavior in animal models of mood disorders
Hodes, Georgia E. (BMC, 2013)
Women have a higher incidence of stress related disorders including depression and generalized anxiety disorder, and epigenetic mechanisms likely contribute to this sex difference. Evidence from preclinical research suggests that epigenetic mechanisms are responsible for both sexual dimorphism of brain regions and sensitivity of the stress response. Epigenetic modifications such as DNA methylation and histone modifications can occur transgenerationally, developmentally, or in response to environmental stimuli such as stress exposure. This review will provide an overview of the various forms of epigenetic modifications observed in the central nervous system and will explain how these mechanisms contribute to a sexually dimorphic brain. It will also discuss the ways in which epigenetic alterations coincide with, and functionally contribute to, the behavioral response to stress across the lifespan. Ultimately, this review will focus on novel research utilizing animal models to investigate sex differences in epigenetic mechanisms that influence susceptibility to stress. Exploration of this relationship reveals epigenetic mechanisms with the potential to explain sexual dimorphism in the occurrence of stress related disorders.
Functional changes in glutamate transporters and astrocyte biophysical properties in a rodent model of focal cortical dysplasia
Campbell, Susan L.; Hablitz, John J.; Olsen, Michelle L. (Frontiers, 2014-12-17)
Cortical dysplasia is associated with intractable epilepsy and developmental delay in young children. Recent work with the rat freeze-induced focal cortical dysplasia (FCD) model has demonstrated that hyperexcitability in the dysplastic cortex is due in part to higher levels of extracellular glutamate. Astrocyte glutamate transporters play a pivotal role in cortical maintaining extracellular glutamate concentrations. Here we examined the function of astrocytic glutamate transporters in a FCD model in rats. Neocortical freeze lesions were made in postnatal day (PN) 1 rat pups and whole cell electrophysiological recordings and biochemical studies were performed at PN 21–28. Synaptically evoked glutamate transporter currents in astrocytes showed a near 10-fold reduction in amplitude compared to sham operated controls. Astrocyte glutamate transporter currents from lesioned animals were also significantly reduced when challenged exogenously applied glutamate. Reduced astrocytic glutamate transport clearance contributed to increased NMDA receptor-mediated current decay kinetics in lesioned animals. The electrophysiological profile of astrocytes in the lesion group was also markedly changed compared to sham operated animals. Control astrocytes demonstrate large-amplitude linear leak currents in response to voltage-steps whereas astrocytes in lesioned animals demonstrated significantly smaller voltage-activated inward and outward currents. Significant decreases in astrocyte resting membrane potential and increases in input resistance were observed in lesioned animals. However, Western blotting, immunohistochemistry and quantitative PCR demonstrated no differences in the expression of the astrocytic glutamate transporter GLT-1 in lesioned animals relative to controls. These data suggest that, in the absence of changes in protein or mRNA expression levels, functional changes in astrocytic glutamate transporters contribute to neuronal hyperexcitability in the FCD model.
A Time-Series Model of Phase Amplitude Cross Frequency Coupling and Comparison of Spectral Characteristics with Neural Data
Lepage, Kyle Q.; Vijayan, Sujith (Hindawi, 2015)
Stochastic processes that exhibit cross-frequency coupling (CFC) are introduced. The ability of these processes to model observed CFC in neural recordings is investigated by comparison with published spectra. One of the proposedmodels, based onmultiplying a pulsatile function of a low-frequency oscillation (𝜃) with an unobserved and high-frequency component, yields a process with a spectrumthat is consistent with observation. Othermodels, such as those employing a biphasic pulsatile function of a low-frequency oscillation, are demonstrated to be less suitable.We introduce the full stochastic process time seriesmodel as a summation of three component weak-sense stationary (WSS) processes, namely, 𝜃, 𝛾, and 𝜂, with 𝜂 a 1/𝑓𝛼 noise process. The 𝛾 process is constructed as a product of a latent and unobserved high-frequency process 𝑥 with a function of the lagged, low-frequency oscillatory component (𝜃). After demonstrating that the model process is WSS, an appropriate method of simulation is introduced based upon the WSS property.This work may be of interest to researchers seeking to connect inhibitory and excitatory dynamics directly to observation in a model that accounts for known temporal dependence or to researchers seeking to examine what can occur in a multiplicative time-domain CFC mechanism.
Novel Applications of Magnetic Cell Sorting to Analyze Cell-Type Specific Gene and Protein Expression in the Central Nervous System
Holt, Leanne M.; Olsen, Michelle L. (PLOS, 2016-02-26)
The isolation and study of cell-specific populations in the central nervous system(CNS) has gained significant interest in the neuroscience community. The ability to examine cell-specific gene and protein expression patterns in healthy and pathological tissue is critical for our understanding of CNS function. Several techniques currently exist to isolate cell-specific populations, each having their own inherent advantages and shortcomings. Isolation of distinct cell populations using magnetic sorting is a technique which has been available for nearly 3 decades, although rarely used in adult whole CNS tissue homogenate. In the current study we demonstrate that distinct cell populations can be isolated in rodents from early postnatal development through adulthood. We found this technique to be amendable to customization using commercially available membrane-targeted antibodies, allowing for cell-specific isolation across development and animal species. This technique yields RNA which can be utilized for downstream applications—including quantitative PCR and RNA sequencing—at relatively low cost and without the need for specialized equipment or fluorescently labeled cells. Adding to its utility, we demonstrate that cells can be isolated largely intact, retaining their processes, enabling analysis of extrasomatic proteins.We propose that magnetic cell sorting will prove to be a highly useful technique for the examination of cell specific CNS populations.
The Ionotropic Receptors IR21a and IR25a mediate cool sensing in Drosophila
Ni, Lina; Klein, Mason; Svec, Kathryn V.; Budelli, Gonzalo; Chang, Elaine C.; Ferrer, Anggie J.; Benton, Richard; Samuel, Aravinthan D. T.; Garrity, Paul A. (eLife Sciences Publications, 2016-04-29)
Animals rely on highly sensitive thermoreceptors to seek out optimal temperatures, but the molecular mechanisms of thermosensing are not well understood. The Dorsal Organ Cool Cells (DOCCs) of the Drosophila larva are a set of exceptionally thermosensitive neurons critical for larval cool avoidance. Here, we show that DOCC cool-sensing is mediated by Ionotropic Receptors (IRs), a family of sensory receptors widely studied in invertebrate chemical sensing. We find that two IRs, IR21a and IR25a, are required to mediate DOCC responses to cooling and are required for cool avoidance behavior. Furthermore, we find that ectopic expression of IR21a can confer coolresponsiveness in an Ir25a-dependent manner, suggesting an instructive role for IR21a in thermosensing. Together, these data show that IR family receptors can function together to mediate thermosensation of exquisite sensitivity.
Neonatal maternal separation stress elicits lasting DNA methylation changes in the hippocampus of stress-reactive Wistar Kyoto rats
McCoy, Chelsea R.; Rana, Samir; Stringfellow, Sara anne; Day, Jeremy; Wyss, J. Michael; Clinton, Sarah M.; Kerman, Ilan A. (Wiley-Blackwell, 2016-11-01)
Early-life stress (ELS) can alter neurodevelopment in variable ways, ranging from producing deleterious outcomes to stress resilience. While most ELS studies focus on its harmful effects, recent work by our lab and others shows that ELS elicits positive effects in certain individuals. We exposed Wistar-Kyoto (WKY) rats, known for a stress reactive, anxiety-/depression-like phenotype, to maternal separation (MS), a model of ELS. MS exposure elicited anxiolytic and antidepressant behavioral effects as well as improved cardiovascular function in adult WKY offspring. The present study interrogates an epigenetic mechanism (DNA methylation) that may confer the adaptive effects of MS in WKY offspring. We quantified global genome methylation levels in limbic brain regions of adult WKYs exposed to daily 180-min MS or neonatal handling from postnatal day 1-14. MS exposure triggered dramatic DNA hypermethylation specifically in the hippocampus. Next-generation sequencing methylome profiling revealed reduced methylation at intragenic sites within two key nodes of insulin signaling pathways: the insulin receptor and one of its major downstream targets, mitogen activated protein kinase kinase kinase 5 (Map3k5). We then tested the hypothesis that enhancing DNA methylation in WKY rats would elicit adaptive changes akin to the effects of MS. Dietary methyl donor supplementation improved WKY rats’ anxiety/depression-like behaviors and also improved cardiovascular measures, similar to previous observations following MS. Overall these data suggest a potential molecular mechanism that mediates a predicted adaptive response whereby ELS induces DNA methylation changes in the brain that may contribute to successful stress coping and adaptive physiological changes in adulthood.
Frontal beta-theta network during REM sleep
Vijayan, Sujith; Lepage, Kyle Q.; Kopell, Nancy J.; Cash, Sydney S. (eLife Sciences Publications, 2017-01-25)
We lack detailed knowledge about the spatio-temporal physiological signatures of REM sleep, especially in humans. By analyzing intracranial electrode data from humans, we demonstrate for the first time that there are prominent beta (15–35 Hz) and theta (4–8 Hz) oscillations in both the anterior cingulate cortex (ACC) and the DLPFC during REM sleep. We further show that these theta and beta activities in the ACC and the DLPFC, two relatively distant but reciprocally connected regions, are coherent. These findings suggest that, counter to current prevailing thought, the DLPFC is active during REM sleep and likely interacting with other areas. Since the DLPFC and the ACC are implicated in memory and emotional regulation, and the ACC has motor areas and is thought to be important for error detection, the dialogue between these two areas could play a role in the regulation of emotions and in procedural motor and emotional memory consolidation.
Differential stress induced c-Fos expression and identification of region-specific miRNA-mRNA networks in the dorsal raphe and amygdala of high-responder/low-responder rats
Cohen, Joshua L.; Ata, Anoosha E.; Jackson, Nateka L.; Rahn, Elizabeth J.; Ramaker, Ryne C.; Cooper, Sara; Kerman, Ilan A.; Clinton, Sarah M. (2017-02)
Chronic stress triggers a variety of physical and mental health problems, and how individuals 2 cope with stress influences risk for emotional disorders. To investigate molecular mechanisms 3 underlying distinct stress coping styles, we utilized rats that were selectively-bred for differences 4 in emotionality and stress reactivity. We show that high novelty responding (HR) rats readily 5 bury a shock probe in the defensive burying test, a measure of proactive stress coping behavior, 6 while low novelty responding (LR) rats exhibit enhanced immobility, a measure of reactive 7 coping. Shock exposure in the defensive burying test elicited greater activation of HR rats’ 8 caudal dorsal raphe serotonergic cells compared to LRs, but lead to more pronounced 9 activation throughout LRs’ amygdala (lateral, basolateral, central, and basomedial nuclei) 10 compared to HRs. RNA-sequencing revealed 271 mRNA transcripts and 33 microRNA species 11 that were differentially expressed in HR/LR raphe and amygdala. We mapped potential 12 microRNA-mRNA networks by correlating and clustering mRNA and microRNA expression and 13 identified networks that differed in either the HR/LR dorsal raphe or amygdala. A dorsal raphe 14 network linked three microRNAs which were down-regulated in LRs (miR-206-3p, miR-3559-5p, 15 and miR-378a-3p) to repression of genes related to microglia and immune response (Cd74, 16 Cyth4, Nckap1l, and Rac2), the genes themselves were up-regulated in LR dorsal raphe. In the 17 amygdala, another network linked miR-124-5p, miR-146a-5p, miR-3068-3p, miR-380-5p, miR-18 539-3p, and miR-7a-1-3p with repression of chromatin remodeling-related genes (Cenpk, 19 Cenpq, Itgb3bp, and Mis18a). Overall this work highlights potential drivers of gene-networks 20 and downstream molecular pathways within the raphe and amygdala that contribute to 21 individual differences in stress coping styles and stress vulnerabilities.
Genetic predisposition to high anxiety- and depression-like behavior coincides with diminished DNA methylation in the adult rat amygdala.
McCoy, Chelsea R.; Jackson, Nateka L.; Day, Jeremy; Clinton, Sarah M. (2017-03-01)
Understanding biological mechanisms that shape vulnerability to emotional dysfunction is critical for elucidating the neurobiology of psychiatric illnesses like anxiety and depression. To elucidate molecular and epigenetic alterations in the brain that contribute to individual differences in emotionality, our laboratory utilized a rodent model of temperamental differences. Rats bred for low response to novelty (Low Responders, LRs) are inhibited in novel situations and display high anxiety, helplessness, and diminished sociability compared to High Novelty Responder (HR) rats. Our current transcriptome profiling experiment identified widespread gene expression differences in the amygdala of adult HR/LR rats; we hypothesize that HR/LR gene expression and downstream behavioral differences stem from distinct epigenetic (specifically DNA methylation) patterning in the HR/LR brain. Although we found similar levels of DNA methyltransferase proteins in the adult HR/LR amygdala, next-generation sequencing analysis of the methylome revealed 793 differentially methylated genomic sites between the groups. Most of the differentially methylated sites were hypermethylated in HR versus LR, so we next tested the hypothesis that enhancing DNA methylation in LRs would improve their anxiety/depression-like phenotype. We found that increasing DNA methylation in LRs (via increased dietary methyl donor content) improved their anxiety-like behavior and decreased their typically high levels of Forced Swim Test (FST) immobility; however, dietary methyl donor depletion exacerbated LRs' high FST immobility. These data are generally consistent with findings in depressed patients showing that treatment with DNA methylation-promoting agents improves depressive symptoms, and highlight epigenetic mechanisms that may contribute to individual differences in risk for emotional dysfunction.
Altered peripheral immune profiles in treatment-resistant depression: response to ketamine and prediction of treatment outcome.
Kiraly, D. D.; Horn, S. R.; Van Dam, N. T.; Costi, S.; Schwartz, J.; Kim-Schulze, S.; Patel, M.; Hodes, Georgia E.; Russo, Scott J.; Merad, Miriam; Iosifescu, D. V.; Charney, D. S.; Murrough, J.W. (2017-03-21)
A subset of patients with depression have elevated levels of inflammatory cytokines, and some studies demonstrate interaction between inflammatory factors and treatment outcome. However, most studies focus on only a narrow subset of factors in a patient sample. In the current study, we analyzed broad immune profiles in blood from patients with treatment-resistant depression (TRD) at baseline and following treatment with the glutamate modulator ketamine. Serum was analyzed from 26 healthy control and 33 actively depressed TRD patients free of antidepressant medication, and matched for age, sex and body mass index. All subjects provided baseline blood samples, and TRD subjects had additional blood draw at 4 and 24 h following intravenous infusion of ketamine (0.5 mg kg-1). Samples underwent multiplex analysis of 41 cytokines, chemokines and growth factors using quantitative immunoassay technology. Our a priori hypothesis was that TRD patients would show elevations in canonical pro-inflammatory cytokines; analyses demonstrated significant elevation of the pro-inflammatory cytokine interleukin-6. Further exploratory analyses revealed significant regulation of four additional soluble factors in patients with TRD. Several cytokines showed transient changes in level after ketamine, but none correlated with treatment response. Low pretreatment levels of fibroblast growth factor 2 were associated with ketamine treatment response. In sum, we found that patients with TRD demonstrate a unique pattern of increased inflammatory mediators, chemokines and colony-stimulating factors, providing support for the immune hypothesis of TRD. These patterns suggest novel treatment targets for the subset of patients with TRD who evidence dysregulated immune functioning.
Positive Modulatory Interactions of NMDA Receptor GluN1/2B Ligand Binding Domains Attenuate Antagonists Activity
Bledsoe, Douglas N.; Tamer, Ceyhun; Mesic, Ivana; Madry, Christian; Klein, Bradley G.; Laube, Bodo; Costa, Blaise M. (Frontiers, 2017-05-09)
N-methyl D-aspartate receptors (NMDAR) play crucial role in normal brain function and pathogenesis of neurodegenerative and psychiatric disorders. Functional tetra-heteromeric NMDAR contains two obligatory GluN1 subunits and two identical or different non-GluN1 subunits that include six different gene products; four GluN2 (A-D) and two GluN3 (A-B) subunits. The heterogeneity of subunit combination facilities the distinct function of NMDARs. All GluN subunits contain an extracellular N-terminal Domain (NTD) and ligand binding domain (LBD), transmembrane domain (TMD) and an intracellular C-terminal domain (CTD). Interaction between the GluN1 and co-assembling GluN2/3 subunits through the LBD has been proven crucial for defining receptor deactivation mechanisms that are unique for each combination of NMDAR. Modulating the LBD interactions has great therapeutic potential. In the present work, by amino acid point mutations and electrophysiology techniques, we have studied the role of LBD interactions in determining the effect of well-characterized pharmacological agents including agonists, competitive antagonists, and allosteric modulators. The results reveal that agonists (glycine and glutamate) potency was altered based on mutant amino acid sidechain chemistry and/or mutation site. Most antagonists inhibited mutant receptors with higher potency; interestingly, clinically used NMDAR channel blocker memantine was about three-fold more potent on mutated receptors (N521A, N521D, and K531A) than wild type receptors. These results provide novel insights on the clinical pharmacology of memantine, which is used for the treatment of mild to moderate Alzheimer's disease. In addition, these findings demonstrate the central role of LBD interactions that can be exploited to develop novel NMDAR based therapeutics.
Zika Virus Persistently and Productively Infects Primary Adult Sensory Neurons In Vitro
Swartwout, Brianna K.; Zlotnick, Marta G.; Saver, Ashley E.; McKenna, Caroline M.; Bertke, Andrea S. (MDPI, 2017-10-13)
Zika virus (ZIKV) has recently surged in human populations, causing an increase in congenital and Guillain-Barré syndromes. While sexual transmission and presence of ZIKV in urine, semen, vaginal secretions, and saliva have been established, the origin of persistent virus shedding into biological secretions is not clear. Using a primary adult murine neuronal culture model, we have determined that ZIKV persistently and productively infects sensory neurons of the trigeminal and dorsal root ganglia, which innervate glands and mucosa of the face and the genitourinary tract, respectively, without apparent injury. Autonomic neurons that innervate these regions are not permissive for infection. However, productive ZIKV infection of satellite glial cells that surround and support sensory and autonomic neurons in peripheral ganglia results in their destruction. Persistent infection of sensory neurons, without affecting their viability, provides a potential reservoir for viral shedding in biological secretions for extended periods of time after infection. Furthermore, viral destruction of satellite glial cells may contribute to the development of Guillain-Barré Syndrome via an alternative mechanism to the established autoimmune response.
Adenosine Signaling through A1 Receptors Inhibits Chemosensitive Neurons in the Retrotrapezoid Nucleus
James, S. D.; Hawkins, V. E.; Falquetto, B.; Ruskin, D. N.; Masino, S. A.; Moreira, T. S.; Olsen, Michelle L.; Mulkey, D. K. (Society for Neuroscience, 2018)
A subset of neurons in the retrotrapezoid nucleus (RTN) function as respiratory chemoreceptors by regulating depth and frequency of breathing in response to changes in tissue CO2/H. The activity of chemosensitive RTN neurons is also subject to modulation by CO2/H-dependent purinergic signaling. However, mechanisms contributing to purinergic regulation of RTN chemoreceptors are not entirely clear. Recent evidence suggests adenosine inhibits RTN chemoreception in vivo by activation of A1 receptors. The goal of this study was to characterize effects of adenosine on chemosensitive RTN neurons and identify intrinsic and synaptic mechanisms underlying this response. Cell-attached recordings from RTN chemoreceptors in slices from rat or wild-type mouse pups (mixed sex) show that exposure to adenosine (1 M) inhibits chemoreceptor activity by an A1 receptor-dependent mechanism. However, exposure to a selective A1 receptor antagonist (8-cyclopentyl-1,3- dipropylxanthine, DPCPX; 30 nM) alone did not potentiate CO2/H-stimulated activity, suggesting activation of A1 receptors does not limit chemoreceptor activity under these reduced conditions. Whole-cell voltage-clamp from chemosensitive RTN neurons shows that exposure to adenosine activated an inward rectifying K conductance, and at the network level, adenosine preferentially decreased frequency of EPSCs but not IPSCs. These results show that adenosine activation of A1 receptors inhibits chemosensitive RTN neurons by direct activation of a G-protein-regulated inward-rectifier K (GIRK)-like conductance, and presynaptically, by suppression of excitatory synaptic input to chemoreceptors.
Dual color optogenetic control of neural populations using low-noise, multishank optoelectrodes
Kampasi, Komal; English, Daniel Fine; Seymour, John; Stark, Eran; McKenzie, Sam; Vöröslakos, Mihály; Buzsáki, György; Wise, Kensall D.; Yoon, Euisik (Nature, 2018)
Optogenetics allows for optical manipulation of neuronal activity and has been increasingly combined with intracellular and extracellular electrophysiological recordings. Genetically-identified classes of neurons are optically manipulated, though the versatility of optogenetics would be increased if independent control of distinct neural populations could be achieved on a sufficient spatial and temporal resolution. We report a scalable multisite optoelectrode design that allows simultaneous optogenetic control of two spatially intermingled neuronal populations in vivo. We describe the design, fabrication, and assembly of low-noise, multisite/multicolor optoelectrodes. Each shank of the four-shank assembly is monolithically integrated with 8 recording sites and a dualcolor waveguide mixer with a 7 × 30 μm cross-section, coupled to 405 nm and 635 nm injection laser diodes (ILDs) via gradient-index (GRIN) lenses to meet optical and thermal design requirements. To better understand noise on the recording channels generated during diode-based activation, we developed a lumped-circuit modeling approach for EMI coupling mechanisms and used it to limit artifacts to amplitudes under 100 μV upto an optical output power of 450 μW. We implanted the packaged devices into the CA1 pyramidal layer of awake mice, expressing Channelrhodopsin-2 in pyramidal cells and ChrimsonR in paravalbumin-expressing interneurons, and achieved optical excitation of each cell type using sub-mW illumination. We highlight the potential use of this technology for functional dissection of neural circuits.
MeCP2 Deficiency Leads to Loss of Glial Kir4.1
Kahanovitch, Uri; Cuddapah, Vishnu A.; Pacheco, Natasha L.; Holt, Leanne M.; Murphy, Daniel K.; Percy, Alan K.; Olsen, Michelle L. (Society for Neuroscience, 2018)
Rett syndrome is a devastating neurodevelopmental disorder that affects 1 in 10,000–25,000 females. Mutations in methyl-CpG-binding protein 2 (MeCP2), a transcriptional regulator, are responsible for >95% of RTT cases. Recent work has shown that astrocytes contribute significantly to the disorder, although their contribution to this disease is not known. Here, we demonstrate that the critical astrocyte K⁺ channel Kir4.1 is a novel molecular target of MeCP2. MeCP2 deficiency leads to decreased Kcnj10/Kir4.1 mRNA levels, protein expression, and currents. These findings provide novel mechanistic insight and begin to elucidate the role of astrocytes in this disorder.

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