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  • 51.
    Johansson, Tobias
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Elfverson, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Zhou, Qin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Nyberg, Fred
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Allosteric modulation of the NMDA receptor by neurosteroids in rat brain and the impact of long term morphine administration2010In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 401, no 4, p. 504-508Article in journal (Refereed)
    Abstract [en]

    This study examined the allosteric modulation of the NMDA receptor by nanomolar concentrations of neurosteroids in rats treated long term with morphine The neurosteroids dehydroepiandrosterone sulfate (DHEAS) pregnenolone sulfate (PS) and pregnanolone sulfate (3 alpha 5 beta S) are important mediators in the central nervous system They induce rapid responses by non-classical steroidal mechanisms e g via interaction with the N-methyl-D-aspartate (NMDA) receptor and are known to modify the binding of ifenprodil to the NMDA receptor subunit NR2B The NMDA receptor is involved in several processes including memory learning synaptic plasticity and neuronal development Morphine a mu-opioid receptor agonist has an important role in the clinical treatment of pain The main drawback of morphine treatment is the associated development of dependence and tolerance The mechanisms behind these phenomena are still to be elucidated but several reports suggest the involvement of the NMDA receptor The results of the present study indicate that the allosteric modulation induced by the neurosteroids DHEAS PS and 3 alpha 5 beta s was similar in all tested brain regions This suggests that the NR2B receptor subunit behaves independently of its site of expression Moreover the NR2B subunit was up-regulated in the frontal cortex but not in the hippocampus or hypothalamus It is concluded that morphine does not affect the neurosteroid modulatory effect on ifenprodil binding in the rat hippocampus or hypothalamus but does significantly affect both the expression of the NR2B subunit and the 3 alpha 3 beta S modulatory effect on ifenprodil binding in the frontal cortex It is suggested that the observed effect of long term morphine on the properties of NR2B in the frontal cortex may be associated with the mechanism underlying the development of opiate dependence.

  • 52.
    Kullander, Klas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Genetics moving to neuronal networks2005In: TINS - Trends in Neurosciences, ISSN 0166-2236, E-ISSN 1878-108X, Vol. 28, no 5, p. 239-247Article in journal (Refereed)
    Abstract [en]

    Neuronal circuits are essential components of the nervous system and determine various body functions. To understand how neuronal circuits operate it is necessary to identify the participating neuronal subpopulations and to dissect the function of the neurons at the molecular level. The locomotor central pattern generator that coordinates body movements is well suited for elucidating the assembly and identity of the participating neurons. Remarkable advances in the field of genetics are making studies in neuroscience more efficient and precise so that now, using nematode worms, fruit flies, zebrafish and mice as model organisms, a genetic approach can be used to identify molecules and neurons crucial for locomotor network functionality.

  • 53.
    Kullander, Klas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Genetics moving to neuronal networks2005In: TINS - Trends in Neurosciences, ISSN 0166-2236, E-ISSN 1878-108X, Vol. 28, no 5, p. 239-247Article in journal (Refereed)
    Abstract [en]

    Neuronal circuits are essential components of the nervous system and determine various body functions. To understand how neuronal circuits operate it is necessary to identify the participating neuronal subpopulations and to dissect the function of the neurons at the molecular level. The locomotor central pattern generator that coordinates body movements is well suited for elucidating the assembly and identity of the participating neurons. Remarkable advances in the field of genetics are making studies in neuroscience more efficient and precise so that now, using nematode worms, fruit flies, zebrafish and mice as model organisms, a genetic approach can be used to identify molecules and neurons crucial for locomotor network functionality.

  • 54.
    Lagerström, Malin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Vglut2-Regulated Itch - Primary Afferents And Beyond2013In: Acta Dermato-Venereologica, ISSN 0001-5555, E-ISSN 1651-2057, Vol. 93, no 5, p. 607-607Article in journal (Other academic)
  • 55.
    Lagerström, Malin C.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Rabe, Nadine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Haitina, Tatjana
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Kalnina, Ineta
    Hellström, Anders R.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Klovins, Janis
    Kullander, Klas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Schiöth, Helgi B.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    The evolutionary history and tissue mapping of GPR123: specific CNS expression pattern predominantly in thalamic nuclei and regions containing large pyramidal cells2007In: Journal of Neurochemistry, ISSN 0022-3042, E-ISSN 1471-4159, Vol. 100, no 4, p. 1129-1142Article in journal (Refereed)
    Abstract [en]

    The Adhesion family of G protein-coupled receptors (GPCRs) includes 33 receptors and is the second largest GPCR family. Most of these proteins are still orphans and fairly little is known of their tissue distribution and evolutionary context. We report the evolutionary history of the Adhesion family protein GPR123 as well as mapping of GPR123 mRNA expression in mouse and rat using in situ hybridization and real-time PCR, respectively. GPR123 was found to be well conserved within the vertebrate lineage, especially within the transmembrane regions and in the distal part of the cytoplasmic tail, containing a potential PDZ binding domain. The real-time PCR data indicates that GPR123 is predominantly expressed in CNS. The in situ data show high expression in thalamic nuclei and regions containing large pyramidal cells like cortex layers 5 and 6 and subiculum. Moreover, we found distinct expression in amygdala, hypothalamus, inferior olive and spinal cord. The CNS specific expression, together with the high sequence conservation between the vertebrate sequences investigated, indicate that GPR123 may have an important role in the regulation of neuronal signal transduction.

  • 56.
    Lagerström, Malin C.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Rogoz, Katarzyna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Abrahamsen, Bjarke
    Lind, Anne-Li
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Ölund, Caroline
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Smith, Casey
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Mendez, José Alfredo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Wallén-Mackenzie, Åsa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Wood, John N.
    Kullander, Klas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    A sensory subpopulation depends on vesicular glutamate transporter 2 for mechanical pain, and together with substance P, inflammatory pain2011In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 108, no 14, p. 5789-5794Article in journal (Refereed)
    Abstract [en]

    Ablating or functionally compromising sets of sensory neurons has provided important insights into peripheral modality-specific wiring in the somatosensory system. Inflammatory hyperalgesia, cold pain, and noxious mechanosensation have all been shown to depend upon Na(v)1.8-positive sensory neurons. The release of fast-acting neurotransmitters, such as glutamate, and more slowly released neuropeptides, such as substance P (SP), contribute to the diversified responses to external stimuli. Here we show that deleting Vglut2 in Na(v)1.8(Cre)-positive neurons compromised mechanical pain and NGF-induced thermal hyperalgesia, whereas tactile-evoked sensation, thermal, formalin-evoked, and chronic neuropathic pain were normal. However, when Vglut2(f/f); Na(v)1.8(Cre) mice were injected with a SP antagonist before the formalin test, the second phase pain response was nearly completely abolished, whereas in control mice, the pain response was unaffected. Our results suggest that VGLUT2-dependent signaling originating from Na(v)1.8-positive neurons is a principal sensing mechanism for mechanical pain and, together with SP, inflammatory pain. These data define sets of primary afferents associated with specific modalities and provide useful genetic tools with which to analyze the pathways that are activated by functionally distinct neuronal populations and transmitters.

  • 57.
    Lagerström, Malin C.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Rogoz, Katarzyna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Abrahamsen, Bjarke
    Persson, Emma
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Reinius, Björn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Nordenankar, Karin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Ölund, Caroline
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Smith, Casey
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Mendez, José Alfredo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Chen, Zhou-Feng
    Wood, John N.
    Wallén-Mackenzie, Åsa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Kullander, Klas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    VGLUT2-Dependent Sensory Neurons in the TRPV1 Population Regulate Pain and Itch2010In: Neuron, ISSN 0896-6273, E-ISSN 1097-4199, Vol. 68, no 3, p. 529-542Article in journal (Refereed)
    Abstract [en]

    The natural response to itch sensation is to scratch, which relieves the itch through an unknown mechanism. Interaction between pain and itch has been frequently demonstrated, and the selectivity hypothesis of itch, based on data from electrophysiological and behavioral experiments, postulates the existence of primary pain afferents capable of repressing itch. Here, we demonstrate that deletion of vesicular glutamate transporter (VGLUT) 2 in a subpopulation of neurons partly overlapping with the vanilloid receptor (TRPV1) primary afferents resulted in a dramatic increase in itch behavior accompanied by a reduced responsiveness to thermal pain. The increased itch behavior was reduced by administration of antihistaminergic drugs and by genetic deletion of the gastrin-releasing peptide receptor, demonstrating a dependence on VGLUT2 to maintain normal levels of both histaminergic and nonhistaminergic itch. This study establishes that VGLUT2 is a major player in TRPV1 thermal nociception and also serves to regulate a normal itch response.

  • 58. Langer, Dominik
    et al.
    van 't Hoff, Marcel
    Keller, Andreas J.
    Nagaraja, Chetan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Pfaeffli, Oliver A.
    Goeldi, Maurice
    Kasper, Hansjoerg
    Helmchen, Fritjof
    HelioScan: A software framework for controlling in vivo microscopy setups with high hardware flexibility, functional diversity and extendibility2013In: Journal of Neuroscience Methods, ISSN 0165-0270, E-ISSN 1872-678X, Vol. 215, no 1, p. 38-52Article in journal (Refereed)
    Abstract [en]

    Intravital microscopy such as in vivo imaging of brain dynamics is often performed with custom-built microscope setups controlled by custom-written software to meet specific requirements. Continuous technological advancement in the field has created a need for new control software that is flexible enough to support the biological researcher with innovative imaging techniques and provide the developer with a solid platform for quickly and easily implementing new extensions. Here, we introduce HelioScan, a software package written in LabVIEW, as a platform serving this dual role. HelioScan is designed as a collection of components that can be flexibly assembled into microscope control software tailored to the particular hardware and functionality requirements. Moreover, HelioScan provides a software framework, within which new functionality can be implemented in a quick and structured manner. A specific HelioScan application assembles at run-time from individual software components, based on user-definable configuration files. Due to its component-based architecture, HelioScan can exploit synergies of multiple developers working in parallel on different components in a community effort. We exemplify the capabilities and versatility of HelioScan by demonstrating several in vivo brain imaging modes, including camera-based intrinsic optical signal imaging for functional mapping of cortical areas, standard two-photon laser-scanning microscopy using galvanometric mirrors, and high-speed in vivo two-photon calcium imaging using either acousto-optic deflectors or a resonant scanner. We recommend HelioScan as a convenient software framework for the in vivo imaging community.

  • 59.
    Larhammar, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Neuronal Networks of Movement: Slc10a4 as a Modulator & Dmrt3 as a Gait-keeper2014Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Nerve cells are organized into complex networks that comprise the building blocks of our nervous system. Neurons communicate by transmitting messenger molecules released from synaptic vesicles. Alterations in neuronal circuitry and synaptic signaling contribute to a wide range of neurological conditions, often with consequences for movement. Intrinsic neuronal networks in the spinal cord serve to coordinate vital rhythmic motor functions. In spite of extensive efforts to address the organization of these neural circuits, much remains to be revealed regarding the identity and function of specific interneuron cell types and how neuromodulation tune network activity. In this thesis, two novel genes initially identified as markers for spinal neuronal populations were investigated: Slc10a4 and Dmrt3.

    The orphan transporter SLC10A4 was found to be expressed on synaptic vesicles of the cholinergic system, including motor neurons, as well as in the monoaminergic system, including dopaminergic, serotonergic and noradrenergic nuclei. Thus, it constitutes a novel molecular denominator shared by these classic neuromodulatory systems. SLC10A4 was found to influence vesicular transport of dopamine and affect neuronal release and reuptake efficiency in the striatum. Mice lacking Slc10a4 displayed impaired monoamine homeostasis and were hypersensitive to the drugs amphetamine and tranylcypromine. These findings demonstrate that SLC10A4 is capable of modulating the modulatory systems of the brain with potential clinical relevance for neurological and mental disorders.

    The transcription factor encoded by Dmrt3 was found to be expressed in a population of inhibitory commissural interneurons originating from the dorsal interneuron 6 (dI6) domain in the spinal cord. In parallel, a genome-wide association study revealed that a non-sense mutation in horse DMRT3 is permissive for the ability to perform pace among other alternate gaits. Further analysis of Dmrt3 null mutant mice showed that Dmrt3 has a central role for spinal neuronal network development with consequences for locomotor behavior. The dI6 class has been suggested to take part in motor circuits but remains one of the least studied classes due to lack of molecular markers. To further investigate the Dmrt3-derived neurons, and the dI6 population in general, a Dmrt3Cre mouse line was generated which allowed for characterization on the molecular, cellular and  behavioral level. It was found that Dmrt3 neurons synapse onto motor neurons, receive extensive synaptic inputs from various neuronal sources and are rhythmically active during fictive locomotion. Furthermore, silencing of Dmrt3 neurons in Dmrt3Cre;Viaatlx/lx mice led to impaired motor coordination and alterations in gait, together demonstrating the importance of this neuronal population in the control of movement.

    List of papers
    1. SLC10A4 Is a Vesicular Amine-Associated Transporter Modulating Dopamine Homeostasis
    Open this publication in new window or tab >>SLC10A4 Is a Vesicular Amine-Associated Transporter Modulating Dopamine Homeostasis
    Show others...
    2015 (English)In: Biological Psychiatry, ISSN 0006-3223, E-ISSN 1873-2402, Vol. 77, no 6, p. 526-536Article in journal (Refereed) Published
    Abstract [en]

    Background

    The neuromodulatory transmitters, biogenic amines, have profound effects on multiple neurons and are essential for normal behavior and mental health. Here we report that the orphan transporter SLC10A4, which in the brain is exclusively expressed in presynaptic vesicles of monoaminergic and cholinergic neurons, has a regulatory role in dopamine homeostasis.

    Methods

    We used a combination of molecular and behavioral analyses, pharmacology, and in vivo amperometry to assess the role of SLC10A4 in dopamine-regulated behaviors.

    Results

    We show that SLC10A4 is localized on the same synaptic vesicles as either vesicular acetylcholine transporter or vesicular monoamine transporter 2. We did not find evidence for direct transport of dopamine by SLC10A4; however, synaptic vesicle preparations lacking SLC10A4 showed decreased dopamine vesicular uptake efficiency. Furthermore, we observed an increased acidification in synaptic vesicles isolated from mice overexpressing SLC10A4. Loss of SLC10A4 in mice resulted in reduced striatal serotonin, noradrenaline, and dopamine concentrations and a significantly higher dopamine turnover ratio. Absence of SLC10A4 led to slower dopamine clearance rates in vivo, which resulted in accumulation of extracellular dopamine. Finally, whereas SLC10A4 null mutant mice were slightly hypoactive, they displayed hypersensitivity to administration of amphetamine and tranylcypromine.

    Conclusions

    Our results demonstrate that SLC10A4 is a vesicular monoaminergic and cholinergic associated transporter that is important for dopamine homeostasis and neuromodulation in vivo. The discovery of SLC10A4 and its role in dopaminergic signaling reveals a novel mechanism for neuromodulation and represents an unexplored target for the treatment of neurological and mental disorders.

    Keywords
    Acetylcholine, amphetamine, central nervous system, cocaine, noradrenaline, serotonin, synaptic transmission, transmitter
    National Category
    Neurosciences Basic Medicine
    Research subject
    Neuroscience
    Identifiers
    urn:nbn:se:uu:diva-214126 (URN)10.1016/j.biopsych.2014.07.017 (DOI)000349776000006 ()25176177 (PubMedID)
    Available from: 2014-01-07 Created: 2014-01-07 Last updated: 2018-01-11
    2. Mutations in DMRT3 affect locomotion in horses and spinal circuit function in mice
    Open this publication in new window or tab >>Mutations in DMRT3 affect locomotion in horses and spinal circuit function in mice
    Show others...
    2012 (English)In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 488, no 7413, p. 642-646Article in journal (Refereed) Published
    Abstract [en]

    Locomotion in mammals relies on a central pattern-generating circuitry of spinal interneurons established during development that coordinates limb movement(1). These networks produce left-right alternation of limbs as well as coordinated activation of flexor and extensor muscles(2). Here we show that a premature stop codon in the DMRT3 gene has a major effect on the pattern of locomotion in horses. The mutation is permissive for the ability to perform alternate gaits and has a favourable effect on harness racing performance. Examination of wild-type and Dmrt3-null mice demonstrates that Dmrt3 is expressed in the dI6 subdivision of spinal cord neurons, takes part in neuronal specification within this subdivision, and is critical for the normal development of a coordinated locomotor network controlling limb movements. Our discovery positions Dmrt3 in a pivotal role for configuring the spinal circuits controlling stride in vertebrates. The DMRT3 mutation has had a major effect on the diversification of the domestic horse, as the altered gait characteristics of a number of breeds apparently require this mutation.

    National Category
    Biological Sciences
    Identifiers
    urn:nbn:se:uu:diva-181404 (URN)10.1038/nature11399 (DOI)000308095100053 ()
    Available from: 2012-09-28 Created: 2012-09-24 Last updated: 2017-12-07Bibliographically approved
    3. Dmrt3 derived neurons are “Gait-keepers” in spinal locomotor circuitry
    Open this publication in new window or tab >>Dmrt3 derived neurons are “Gait-keepers” in spinal locomotor circuitry
    Show others...
    (English)Manuscript (preprint) (Other academic)
    National Category
    Neurosciences
    Research subject
    Neuroscience
    Identifiers
    urn:nbn:se:uu:diva-230429 (URN)
    Projects
    Neuronal Networks of Movement
    Available from: 2014-08-25 Created: 2014-08-25 Last updated: 2018-10-29
  • 60.
    Larhammar, Martin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Nagaraja, Chetan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Perry, Sharn
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Tafreshiha, Atieh
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Hilscher, Markus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Rogoz, Katarzyna
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Restrepo, Ernesto
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Kullander, Klas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Dmrt3 derived neurons are “Gait-keepers” in spinal locomotor circuitryManuscript (preprint) (Other academic)
  • 61.
    Larhammar, Martin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Patra, Kalicharan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Blunder, Martina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Emilsson, Lina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Peuckert, Christiane
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Arvidsson, Emma
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Rönnlund, Daniel
    Royal Institute of Technology, Stockholm.
    Preobraschenski, Julia
    Max Planck Institute for Biophysical Chemistry, Gottingen.
    Birgner, Carolina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Limbach, Christoph
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Widengren, Jerker
    Royal Institute of Technology.
    Blom, Hans
    Royal Institute of Technology.
    Jahn, Reinhard
    Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, 37077 Göttingen, Germany.
    Wallén-Mackenzie, Åsa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Kullander, Klas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    SLC10A4 Is a Vesicular Amine-Associated Transporter Modulating Dopamine Homeostasis2015In: Biological Psychiatry, ISSN 0006-3223, E-ISSN 1873-2402, Vol. 77, no 6, p. 526-536Article in journal (Refereed)
    Abstract [en]

    Background

    The neuromodulatory transmitters, biogenic amines, have profound effects on multiple neurons and are essential for normal behavior and mental health. Here we report that the orphan transporter SLC10A4, which in the brain is exclusively expressed in presynaptic vesicles of monoaminergic and cholinergic neurons, has a regulatory role in dopamine homeostasis.

    Methods

    We used a combination of molecular and behavioral analyses, pharmacology, and in vivo amperometry to assess the role of SLC10A4 in dopamine-regulated behaviors.

    Results

    We show that SLC10A4 is localized on the same synaptic vesicles as either vesicular acetylcholine transporter or vesicular monoamine transporter 2. We did not find evidence for direct transport of dopamine by SLC10A4; however, synaptic vesicle preparations lacking SLC10A4 showed decreased dopamine vesicular uptake efficiency. Furthermore, we observed an increased acidification in synaptic vesicles isolated from mice overexpressing SLC10A4. Loss of SLC10A4 in mice resulted in reduced striatal serotonin, noradrenaline, and dopamine concentrations and a significantly higher dopamine turnover ratio. Absence of SLC10A4 led to slower dopamine clearance rates in vivo, which resulted in accumulation of extracellular dopamine. Finally, whereas SLC10A4 null mutant mice were slightly hypoactive, they displayed hypersensitivity to administration of amphetamine and tranylcypromine.

    Conclusions

    Our results demonstrate that SLC10A4 is a vesicular monoaminergic and cholinergic associated transporter that is important for dopamine homeostasis and neuromodulation in vivo. The discovery of SLC10A4 and its role in dopaminergic signaling reveals a novel mechanism for neuromodulation and represents an unexplored target for the treatment of neurological and mental disorders.

  • 62. Leao, Richardson N.
    et al.
    Reis, Amilcar
    Emirandetti, Amanda
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Lewicka, Michalina
    Hermanson, Ola
    Fisahn, André
    A Voltage-Sensitive Dye-Based Assay for the Identification of Differentiated Neurons Derived from Embryonic Neural Stem Cell Cultures2010In: PLOS ONE, ISSN 1932-6203, Vol. 5, no 11, p. e13833-Article in journal (Refereed)
    Abstract [en]

    Background: Pluripotent and multipotent stem cells hold great therapeutical promise for the replacement of degenerated tissue in neurological diseases. To fulfill that promise we have to understand the mechanisms underlying the differentiation of multipotent cells into specific types of neurons. Embryonic stem cell (ESC) and embryonic neural stem cell (NSC) cultures provide a valuable tool to study the processes of neural differentiation, which can be assessed using immunohistochemistry, gene expression, Ca2+-imaging or electrophysiology. However, indirect methods such as protein and gene analysis cannot provide direct evidence of neuronal functionality. In contrast, direct methods such as electrophysiological techniques are well suited to produce direct evidence of neural functionality but are limited to the study of a few cells on a culture plate. Methodology/Principal Findings: In this study we describe a novel method for the detection of action potential-capable neurons differentiated from embryonic NSC cultures using fast voltage-sensitive dyes (VSD). We found that the use of extracellularly applied VSD resulted in a more detailed labeling of cellular processes compared to calcium indicators. In addition, VSD changes in fluorescence translated precisely to action potential kinetics as assessed by the injection of simulated slow and fast sodium currents using the dynamic clamp technique. We further demonstrate the use of a finite element model of the NSC culture cover slip for optimizing electrical stimulation parameters. Conclusions/Significance: Our method allows for a repeatable fast and accurate stimulation of neurons derived from stem cell cultures to assess their differentiation state, which is capable of monitoring large amounts of cells without harming the overall culture.

  • 63.
    Leao, Richardson N
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Targino, Zé Henrique
    Colom, Luis V
    Fisahn, André
    Interconnection and Synchronization of Neuronal Populations in the Mouse Medial Septum/Diagonal Band of Brocca2015In: Journal of Neurophysiology, ISSN 0022-3077, E-ISSN 1522-1598, Vol. 113, no 3, p. 971-980Article in journal (Refereed)
    Abstract [en]

    The medial septum/diagonal band of Broca (MS/DBB) is crucial for hippocampal theta rhythm generation (4-12Hz). However, the mechanisms behind theta rhythmogenesis are still under debate. The MS/DBB consists, in its majority, of three neuronal populations that use either acetylcholine, GABA or glutamate as neurotransmitters. While the firing patterns of septal neurons enable the MS/DBB to generate rhythmic output critical for the generation of the hippocampal theta rhythm, the ability to synchronize these action potentials is dependent on the interconnectivity between the three major MS/DBB neuronal populations. Yet little is known about intraseptal connections. Here we assess the connectivity between pairs of MS/DBB neurons using paired patch-clamp recordings. We found that glutamatergic and GABAergic neurons provide intraseptal connections and produce sizable currents in MS/DBB postsynaptic cells. We also analyzed linear and non-linear relationships between the action potentials fired by pairs of neurons belonging to various MS/DBB neuronal populations. Our results show that while the synchrony index for action potential firing was significantly higher in pairs of GABAergic neurons coherence of action potential firing in the theta range was similarly low in all pairs analyzed. Recurrence analysis demonstrated that individual action potentials were more recurrent in cholinergic neurons than other cell types. Implementing sparse connectivity in a computer model of the MS/DBB network reproduced our experimental data. We conclude that the interplay between the intrinsic membrane properties of different MS/DBB neuronal populations and the connectivity amongst these populations underlies the ability of the MS/DBB network to critically contribute to hippocampal theta rhythmogenesis.

  • 64.
    Leão, Katarina E.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Leão, Richardson N.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Walmsley, Bruce
    Modulation of dendritic synaptic processing in the lateral superior olive by hyperpolarization-activated currents2011In: European Journal of Neuroscience, ISSN 0953-816X, E-ISSN 1460-9568, Vol. 33, no 8, p. 1462-1470Article in journal (Refereed)
    Abstract [en]

    We have previously shown that mice lateral superior olive (LSO) neurons exhibit a large hyperpolarization-activated current (I(h) ), and that hyperpolarization-activated cyclic-nucleotide-gated type 1 channels are present in both the soma and dendrites of these cells. Here we show that the dendritic I(h) in LSO neurons modulates the integration of multiple synaptic inputs. We tested the LSO neuron's ability to integrate synaptic inputs by evoking excitatory post-synaptic potentials (EPSPs) in conjunction with brief depolarizing current pulses (to simulate a second excitatory input) at different time delays. We compared LSO neurons with the native I(h) present in both the soma and dendrites (control) with LSO neurons without I(h) (blocked with ZD7288) and with LSO neurons with I(h) only present peri-somatically (ZD7288+ computer-simulated I(h) using a dynamic clamp). LSO neurons without I(h) had a wider time window for firing in response to inputs with short time separations. Simulated somatic I(h) (dynamic clamp) could not reverse this effect. Blocking I(h) also increased the summation of EPSPs elicited at both proximal and distal dendritic regions, and dramatically altered the integration of EPSPs and inhibitory post-synaptic potentials. The addition of simulated peri-somatic I(h) could not abolish a ZD7288-induced increase of responsiveness to widely separated excitatory inputs. Using a compartmental LSO model, we show that dendritic I(h) can reduce EPSP integration by locally decreasing the input resistance. Our results suggest a significant role for dendritic I(h) in LSO neurons, where the activation/deactivation of I(h) can alter the LSO response to synaptic inputs.

  • 65.
    Leão, Richardson N.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Colom, Luis V.
    Borgius, Lotta
    Kiehn, Ole
    Fisahn, Andre
    Medial septal dysfunction by A beta-induced KCNQ channel-block in glutamatergic neurons2012In: Neurobiology of Aging, ISSN 0197-4580, E-ISSN 1558-1497, Vol. 33, no 9, p. 2046-2061Article in journal (Refereed)
    Abstract [en]

    Amyloid beta (A beta) peptides play a central role in the pathophysiology of Alzheimer's disease (AD). The cellular mechanisms underlying A beta toxicity, however, are poorly understood. Here we show that A beta 25-35 and A beta 1-40 acutely and differentially affect the characteristics of 3 classes of medial septum (MS) neurons in mice. In glutamatergic neurons A beta increases firing frequency and blocks the A-and the M-current (I-A and I-M, respectively). While the I-A block is similar in other MS neuron classes, the block of I-M is specific to glutamatergic neurons. I-M block and a simulated A beta block mimic the A beta-induced increase in spontaneous firing in glutamatergic neurons. Calcium imaging shows that under control conditions glutamatergic neurons rarely fire while nonglutamatergic neurons fire coherently at theta frequencies. A beta increases the firing rate of glutamatergic neurons while nonglutamatergic neurons lose theta firing coherence. Our results demonstrate that A beta-induced dysfunction of glutamatergic neurons via I-M decrease diminishes MS rhythmicity, which may negatively affect hippocampal rhythmogenesis and underlie the memory loss observed in Alzheimer's disease.

  • 66.
    Leão, Richardson Naves
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Mikulovic, Sanja
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Leão, Katarina E.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Munguba, H.
    Gezelius, Henrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Enjin, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Patra, Kalicharan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Eriksson, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Loew, L. M.
    Tort, A. B. L.
    Kullander, Klas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics. Uppsala University, Science for Life Laboratory, SciLifeLab.
    OLM interneurons differentially modulate CA3 and entorhinal inputs to hippocampal CA1 neurons2012In: Nature Neuroscience, ISSN 1097-6256, E-ISSN 1546-1726, Vol. 15, no 11, p. 1524-1530Article in journal (Refereed)
    Abstract [en]

    The vast diversity of GABAergic interneurons is believed to endow hippocampal microcircuits with the required flexibility for memory encoding and retrieval. However, dissection of the functional roles of defined interneuron types has been hampered by the lack of cell-specific tools. We identified a precise molecular marker for a population of hippocampal GABAergic interneurons known as oriens lacunosum-moleculare (OLM) cells. By combining transgenic mice and optogenetic tools, we found that OLM cells are important for gating the information flow in CA1, facilitating the transmission of intrahippocampal information (from CA3) while reducing the influence of extrahippocampal inputs (from the entorhinal cortex). Furthermore, we found that OLM cells were interconnected by gap junctions, received direct cholinergic inputs from subcortical afferents and accounted for the effect of nicotine on synaptic plasticity of the Schaffer collateral pathway. Our results suggest that acetylcholine acting through OLM cells can control the mnemonic processes executed by the hippocampus.

  • 67.
    Lima da Cruz, Rafael Vitor
    et al.
    Univ Fed Rio Grande do Norte, Brain Inst, Neurodynam Lab, Natal, RN, Brazil.
    Moulin, Thiago C.
    Univ Fed Rio de Janeiro, Inst Med Biochem, Rio De Janeiro, Brazil.
    Petiz, Lyvia Lintzmaier
    Univ Fed Rio Grande do Norte, Brain Inst, Neurodynam Lab, Natal, RN, Brazil.
    Leão, Richardson N
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics. Univ Fed Rio Grande do Norte, Brain Inst, Neurodynam Lab, Natal, RN, Brazil.
    A Single Dose of 5-MeO-DMT Stimulates Cell Proliferation, Neuronal Survivability, Morphological and Functional Changes in Adult Mice Ventral Dentate Gyrus2018In: Frontiers in Molecular Neuroscience, ISSN 1662-5099, Vol. 11, article id 312Article in journal (Refereed)
    Abstract [en]

    The subgranular zone (SGZ) of dentate gyrus (DG) is one of the few regions in which neurogenesis is maintained throughout adulthood. It is believed that newborn neurons in this region encode temporal information about partially overlapping contextual memories. The 5-Methoxy-N,N-dimethyltryptamine (5-MeO-DMT) is a naturally occurring compound capable of inducing a powerful psychedelic state. Recently, it has been suggested that DMT analogs may be used in the treatment of mood disorders. Due to the strong link between altered neurogenesis and mood disorders, we tested whether 5-MeO-DMT is capable of increasing DG cell proliferation. We show that a single intracerebroventricular (ICV) injection of 5-MeO-DMT increases the number of Bromodeoxyuridine (BrdU+) cells in adult mice DG. Moreover, using a transgenic animal expressing tamoxifen-dependent Cre recombinase under doublecortin promoter, we found that 5 Meo-DMT treated mice had a higher number of newborn DG Granule cells (GC). We also showed that these DG GC have more complex dendritic morphology after 5-MeO-DMT. Lastly, newborn GC treated with 5-MeO-DMT, display shorter afterhyperpolarization (AHP) potentials and higher action potential (AP) threshold compared. Our findings show that 5-MeO-DMT affects neurogenesis and this effect may contribute to the known antidepressant properties of DMT-derived compounds.

  • 68.
    Magnúsdóttir, Elín Ingibjörg
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Grujic, Mirjana
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Roers, Axel
    Univ Technol Dresden, Inst Immunol, Dresden, Germany.
    Hartmann, Karin
    Univ Lubeck, Dept Dermatol, Lubeck, Germany.
    Pejler, Gunnar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Swedish Univ Agr Sci, Dept Anat Physiol & Biochem, Uppsala, Sweden.
    Lagerström, Malin C.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Mouse mast cells and mast cell proteases do not play a significant role in acute tissue injury pain induced by formalin2018In: Molecular Pain, ISSN 1744-8069, E-ISSN 1744-8069, Vol. 14, article id 1744806918808161Article in journal (Refereed)
    Abstract [en]

    Subcutaneous formalin injections are used as a model for tissue injury-induced pain where formalin induces pain and inflammation indirectly by crosslinking proteins and directly through activation of the transient receptor potential A1 receptor on primary afferents. Activation of primary afferents leads to both central and peripheral release of neurotransmitters. Mast cells are found in close proximity to peripheral sensory nerve endings and express receptors for neurotransmitters released by the primary afferents, contributing to the neuro/immune interface. Mast cell proteases are found in large quantities within mast cell granules and are released continuously in small amounts and upon mast cell activation. They have a wide repertoire of proposed substrates, including Substance P and calcitonin gene-related peptide, but knowledge of their in vivo function is limited. We evaluated the role of mouse mast cell proteases (mMCPs) in tissue injury pain responses induced by formalin, using transgenic mice lacking either mMCP4, mMCP6, or carboxypeptidase A3 (CPA3), or mast cells in their entirety. Further, we investigated the role of mast cells in heat hypersensitivity following a nerve growth factor injection. No statistical difference was observed between the respective mast cell protease knockout lines and wild-type controls in the formalin test. Mast cell deficiency did not have an effect on formalin-induced nociceptive responses nor nerve growth factor-induced heat hypersensitivity. Our data thus show that mMCP4, mMCP6, and CPA3 as well as mast cells as a whole, do not play a significant role in the pain responses associated with acute tissue injury and inflammation in the formalin test. Our data also indicate that mast cells are not essential to heat hypersensitivity induced by nerve growth factor.

  • 69.
    Markljung, Ellen
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Jiang, Lin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Jaffe, Jacob D.
    Mikkelsen, Tarjei S.
    Wallerman, Ola
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Larhammar, Martin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Zhang, Xiaolan
    Wang, Li
    Saenz-Vash, Veronica
    Gnirke, Andreas
    Lindroth, Anders M.
    Barrés, Romain
    Yan, Jie
    Strömberg, Sara
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    De, Sachinandan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Pontén, Fredrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Lander, Eric S.
    Carr, Steven A.
    Zierath, Juleen R.
    Kullander, Klas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Wadelius, Claes
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
    Lindblad-Toh, Kerstin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Andersson, Göran
    Hjälm, Göran
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Andersson, Leif
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    ZBED6, a novel transcription factor derived from a domesticated DNA transposon regulates IGF2 expression and muscle growth2009In: PLoS biology, ISSN 1544-9173, E-ISSN 1545-7885, Vol. 7, no 12, p. e1000256-Article in journal (Refereed)
    Abstract [en]

    A single nucleotide substitution in intron 3 of IGF2 in pigs abrogates a binding site for a repressor and leads to a 3-fold up-regulation of IGF2 in skeletal muscle. The mutation has major effects on muscle growth, size of the heart, and fat deposition. Here, we have identified the repressor and find that the protein, named ZBED6, is previously unknown, specific for placental mammals, and derived from an exapted DNA transposon. Silencing of Zbed6 in mouse C2C12 myoblasts affected Igf2 expression, cell proliferation, wound healing, and myotube formation. Chromatin immunoprecipitation (ChIP) sequencing using C2C12 cells identified about 2,500 ZBED6 binding sites in the genome, and the deduced consensus motif gave a perfect match with the established binding site in Igf2. Genes associated with ZBED6 binding sites showed a highly significant enrichment for certain Gene Ontology classifications, including development and transcriptional regulation. The phenotypic effects in mutant pigs and ZBED6-silenced C2C12 myoblasts, the extreme sequence conservation, its nucleolar localization, the broad tissue distribution, and the many target genes with essential biological functions suggest that ZBED6 is an important transcription factor in placental mammals, affecting development, cell proliferation, and growth.

  • 70.
    Mikulovic, Sanja
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Pupe, Stéfano
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Peixoto, Helotn Maia
    Do Nascimiento, George
    Kullander, Klas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Tort, Adriano
    Leao, Richardson
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    On the photovoltaic effect in localfield potential recordings2016In: Neurophotonics, ISSN 2329-423X, Vol. 3, no 1, article id 015002Article in journal (Refereed)
    Abstract [en]

    ptogenetics allows light activation of genetically defined cell populations and the study of their link to specific brain functions. While it is a powerful method that has revolutionized neuroscience in the last decade, the shortcomings of directly stimulating electrodes and living tissue with light have been poorly characterized. Here, we assessed the photovoltaic effects in local field potential (LFP) recordings of the mouse hippocampus. We found that light leads to several artifacts that resemble genuine LFP features in animals with no opsin expression, such as stereotyped peaks at the power spectrum, phase shifts across different recording channels, coupling between low and high oscillation frequencies, and sharp signal deflections that are detected as spikes. Further, we tested how light stimulation affected hippocampal LFP recordings in mice expressing channelrhodopsin 2 in parvalbumin neurons (PV/ChR2 mice). Genuine oscillatory activity at the frequency of light stimulation could not be separated from light-induced artifacts. In addition, light stimulation in PV/ChR2 mice led to an overall decrease in LFP power. Thus, genuine LFP changes caused by the stimulation of specific cell populations may be intermingled with spurious changes caused by photovoltaic effects. Our data suggest that care should be taken in the interpretation of electrophysiology experiments involving light stimulation.

  • 71.
    Mikulovic, Sanja
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Restrepo, Carlos Ernesto
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Siwani, Samer
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Bauer, Pavol
    Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Division of Scientific Computing. Uppsala University, Disciplinary Domain of Science and Technology, Mathematics and Computer Science, Department of Information Technology, Computational Science.
    Johann, Stefano Pupe
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Tort, Adriano B. L.
    Kullander, Klas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Leão, Richardson N.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Ventral hippocampal OLM cells control type 2 theta oscillations and response to predator odor2018In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 9, p. 3638:1-15, article id 3638Article in journal (Refereed)
  • 72.
    Mikulovic, Sanja
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Restrepo, Ernesto C.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Hilscher, Markus M.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Kullander, Klas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Leao, Richardson N.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Novel markers for OLM interneurons in the hippocampus2015In: Frontiers in Cellular Neuroscience, ISSN 1662-5102, E-ISSN 1662-5102, Vol. 9, article id 201Article in journal (Other academic)
  • 73.
    Nordenankar, Karin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Neurobiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Bergfors, Assar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Neurobiology.
    Wallén-Mackenzie, Åsa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Neurobiology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Targeted deletion of Vglut2 expression in the embryonal telencephalon promotes an anxiolytic phenotype of the adult mouse2015In: Upsala Journal of Medical Sciences, ISSN 0300-9734, E-ISSN 2000-1967, Vol. 120, no 3Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: Anxiety is a natural emotion experienced by all individuals. However, when anxiety becomes excessive, it contributes to the substantial group of anxiety disorders that affect one in three people and thus are among the most common psychiatric disorders. Anxiolysis, the reduction of anxiety, is mediated via several large groups of therapeutical compounds, but the relief is often only temporary, and increased knowledge of the neurobiology underlying anxiety is needed in order to improve future therapies.

    AIM: We previously demonstrated that mice lacking forebrain expression of the Vesicular glutamate transporter 2 (Vglut2) from adolescence showed a strong anxiolytic behaviour as adults. In the current study, we wished to analyse if removal of Vglut2 expression already from mid-gestation of the mouse embryo would give rise to similar anxiolysis in the adult mouse.

    METHODS: We produced transgenic mice lacking Vglut2 from mid-gestation and analysed their affective behaviour, including anxiety, when they had reached adulthood.

    RESULTS: The transgenic mice lacking Vglut2 expression from mid-gestation showed certain signs of anxiolytic behaviour, but this phenotype was not as prominent as when Vglut2 was removed during adolescence.

    CONCLUSION: Our results suggest that both embryonal and adolescent forebrain expression of Vglut2 normally contributes to balancing the level of anxiety. As the neurobiological basis for anxiety is similar across species, our results in mice may help improve the current understanding of the neurocircuitry of anxiety, and hence anxiolysis, also in humans.

  • 74.
    Nordenankar, Karin
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Pharmacology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Smith-Anttila, Casey J A
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Schweizer, Nadine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Viereckel, Thomas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Birgner, Carolina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Mejia-Toiber, Jana
    Morales, Marisela
    Leao, Richardson Naves
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Wallén-Mackenzie, Åsa
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Increased hippocampal excitability and impaired spatial memory function in mice lacking VGLUT2 selectively in neurons defined by tyrosine hydroxylase promoter activity2015In: Brain Structure and Function, ISSN 1863-2653, E-ISSN 1863-2661, Vol. 220, no 4, p. 2171-2190Article in journal (Refereed)
    Abstract [en]

    Three populations of neurons expressing the vesicular glutamate transporter 2 (Vglut2) were recently described in the A10 area of the mouse midbrain, of which two populations were shown to express the gene encoding, the rate-limiting enzyme for catecholamine synthesis, tyrosine hydroxylase (TH).One of these populations ("TH-Vglut2 Class1") also expressed the dopamine transporter (DAT) gene while one did not ("TH-Vglut2 Class2"), and the remaining population did not express TH at all ("Vglut2-only"). TH is known to be expressed by a promoter which shows two phases of activation, a transient one early during embryonal development, and a later one which gives rise to stable endogenous expression of the TH gene. The transient phase is, however, not specific to catecholaminergic neurons, a feature taken to advantage here as it enabled Vglut2 gene targeting within all three A10 populations expressing this gene, thus creating a new conditional knockout. These knockout mice showed impairment in spatial memory function. Electrophysiological analyses revealed a profound alteration of oscillatory activity in the CA3 region of the hippocampus. In addition to identifying a novel role for Vglut2 in hippocampus function, this study points to the need for improved genetic tools for targeting of the diversity of subpopulations of the A10 area.

  • 75.
    Nordström, Karl J V
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Pharmacology.
    Lagerström, Malin C
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Sciences, Clinical Pharmacology. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Wallér, Linn M J
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Pharmacology.
    Fredriksson, Robert
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Pharmacology.
    Schiöth, Helgi B
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Functional Pharmacology.
    The Secretin GPCRs descended from the family of Adhesion GPCRs2009In: Molecular biology and evolution, ISSN 0737-4038, E-ISSN 1537-1719, Vol. 26, no 1, p. 71-84Article in journal (Refereed)
    Abstract [en]

    The Adhesion G-protein-coupled receptors (GPCRs) are the most complex gene family among GPCRs with large genomic size, multiple introns, and a fascinating flora of functional domains, though the evolutionary origin of this family has been obscure. Here we studied the evolution of all class B (7tm2)-related genes, including the Adhesion, Secretin, and Methuselah families of GPCRs with a focus on nine genomes. We found that the cnidarian genome of Nematostella vectensis has a remarkably rich set of Adhesion GPCRs with a broad repertoire of N-terminal domains although this genome did not have any Secretin GPCRs. Moreover, the single-celled and colony-forming eukaryotes Monosiga brevicollis and Dictyostelium discoideum contain Adhesion-like GPCRs although these genomes do not have any Secretin GPCRs suggesting that the Adhesion types of GPCRs are the most ancient among class B GPCRs. Phylogenetic analysis found Adhesion group V (that contains GPR133 and GPR144) to be the closest relative to the Secretin family in the Adhesion family. Moreover, Adhesion group V sequences in N. vectensis share the same splice site setup as the Secretin GPCRs. Additionally, one of the most conserved motifs in the entire Secretin family is only found in group V of the Adhesion family. We suggest therefore that the Secretin family of GPCRs could have descended from group V Adhesion GPCRs. We found a set of unique Adhesion-like GPCRs in N. vectensis that have long N-termini containing one Somatomedin B domain each, which is a domain configuration similar to that of a set of Adhesion-like GPCRs found in Branchiostoma floridae. These sequences show slight similarities to Methuselah sequences found in insects. The extended class B GPCRs have a very complex evolutionary history with several species-specific expansions, and we identified at least 31 unique N-terminal domains originating from other protein classes. The overall N-terminal domain structure, however, concurs with the phylogenetic analysis of the transmembrane domains, thus enabling us to track the origin of most of the subgroups.

  • 76. Passante, Lara
    et al.
    Gaspard, Nicolas
    Degraeve, Mélanie
    Frisén, Jonas
    Kullander, Klas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    De Maertelaer, Viviane
    Vanderhaeghen, Pierre
    Temporal regulation of ephrin/Eph signalling is required for the spatial patterning of the mammalian striatum2008In: Development, ISSN 0950-1991, E-ISSN 1477-9129, Vol. 135, no 19, p. 3281-3290Article in journal (Refereed)
    Abstract [en]

    Brain structures, whether mature or developing, display a wide diversity of pattern and shape, such as layers, nuclei or segments. The striatum in the mammalian forebrain displays a unique mosaic organization (subdivided into two morphologically and functionally defined neuronal compartments: the matrix and the striosomes) that underlies important functional features of the basal ganglia. Matrix and striosome neurons are generated sequentially during embryonic development, and segregate from each other to form a mosaic of distinct compartments. However, the molecular mechanisms that underlie this time-dependent process of neuronal segregation remain largely unknown. Using a novel organotypic assay, we identified ephrin/Eph family members as guidance cues that regulate matrix/striosome compartmentalization. We found that EphA4 and its ephrin ligands displayed specific temporal patterns of expression and function that play a significant role in the spatial segregation of matrix and striosome neurons. Analysis of the striatal patterning in ephrin A5/EphA4 mutant mice further revealed the requirement of EphA4 signalling for the proper sorting of matrix and striosome neuronal populations in vivo. These data constitute the first identification of genes involved in striatal compartmentalization, and reveal a novel mechanism by which the temporal control of guidance cues enables neuronal segregation, and thereby the generation of complex cellular patterns in the brain.

  • 77.
    Patra, Kalicharan
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Modulation of Neuronal Functions: the Role of SLC10A42014Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Mental health of a person depends on the correct functioning of the brain. The brain and the spinal cord contain many types of cells, of which one important type are called the neurons. Neurons are special in the way they connect to each other to form large networks. The chemicals called transmitters are packed at the nerve endings into tiny packets called vesicles and when a signal arrives these vesicles fuse immediately to the attached cell surface and release their contents. The role of the synaptic vesicular transporter proteins is to ensure proper packing of transmitter molecules that can be released upon stimulation. Vesicular packing is an important process. The carrier proteins involved in packing work in coordination to determine the amount and type of transmitters to be packed. Missing a carrier protein from the vesicles might lead to improper packing and inaccurate signaliing. These signaling molecules are known for their implications in many psychiatric and neurological disorders like Alzheimer’s disease, Parkinson’s disease, Schizophrenia, and attention deficit to name just a few. 

    How a vesicular transporter can affect the modulatory functions of aminergic neurons is the subject of this thesis. This thesis reports on the effects of the loss of a vesicular orphan transporter. Study I demonstrates the localization of this protein to monoaminergic and cholinergic terminals. It reports the effect of the loss of Slc10A4 on vesicular dopamine uptake, synaptic clearance of dopamine and hypersensitivity of animals to dopamine related psychostimulants. Study I also provides evidence for ATP as a possible ligand for SLC10A4 protein. Study II provides data on the clinical relevance of Slc10A4 in playing a protective role against vulnerability to epilepsy. It reports that loss of Slc10A4 renders the animals hypersensitive to cholinergic drugs. Study III provides a closer look at individual cholinergic synapses at neuromuscular junctions in mice lacking Slc10A4. The structural and electrophysiological properties of the NMJ are found compromised because of the loss of this vesicular protein. Taken together, this thesis presents a SV protein’s perspective of viewing at modulation of synaptic transmission.

    List of papers
    1. SLC10A4 Is a Vesicular Amine-Associated Transporter Modulating Dopamine Homeostasis
    Open this publication in new window or tab >>SLC10A4 Is a Vesicular Amine-Associated Transporter Modulating Dopamine Homeostasis
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    2015 (English)In: Biological Psychiatry, ISSN 0006-3223, E-ISSN 1873-2402, Vol. 77, no 6, p. 526-536Article in journal (Refereed) Published
    Abstract [en]

    Background

    The neuromodulatory transmitters, biogenic amines, have profound effects on multiple neurons and are essential for normal behavior and mental health. Here we report that the orphan transporter SLC10A4, which in the brain is exclusively expressed in presynaptic vesicles of monoaminergic and cholinergic neurons, has a regulatory role in dopamine homeostasis.

    Methods

    We used a combination of molecular and behavioral analyses, pharmacology, and in vivo amperometry to assess the role of SLC10A4 in dopamine-regulated behaviors.

    Results

    We show that SLC10A4 is localized on the same synaptic vesicles as either vesicular acetylcholine transporter or vesicular monoamine transporter 2. We did not find evidence for direct transport of dopamine by SLC10A4; however, synaptic vesicle preparations lacking SLC10A4 showed decreased dopamine vesicular uptake efficiency. Furthermore, we observed an increased acidification in synaptic vesicles isolated from mice overexpressing SLC10A4. Loss of SLC10A4 in mice resulted in reduced striatal serotonin, noradrenaline, and dopamine concentrations and a significantly higher dopamine turnover ratio. Absence of SLC10A4 led to slower dopamine clearance rates in vivo, which resulted in accumulation of extracellular dopamine. Finally, whereas SLC10A4 null mutant mice were slightly hypoactive, they displayed hypersensitivity to administration of amphetamine and tranylcypromine.

    Conclusions

    Our results demonstrate that SLC10A4 is a vesicular monoaminergic and cholinergic associated transporter that is important for dopamine homeostasis and neuromodulation in vivo. The discovery of SLC10A4 and its role in dopaminergic signaling reveals a novel mechanism for neuromodulation and represents an unexplored target for the treatment of neurological and mental disorders.

    Keywords
    Acetylcholine, amphetamine, central nervous system, cocaine, noradrenaline, serotonin, synaptic transmission, transmitter
    National Category
    Neurosciences Basic Medicine
    Research subject
    Neuroscience
    Identifiers
    urn:nbn:se:uu:diva-214126 (URN)10.1016/j.biopsych.2014.07.017 (DOI)000349776000006 ()25176177 (PubMedID)
    Available from: 2014-01-07 Created: 2014-01-07 Last updated: 2018-01-11
    2. The synaptic protein encoded by the gene Slc10A4 suppresses epileptiform activity and regulates sensitivity to cholinergic chemoconvulsants
    Open this publication in new window or tab >>The synaptic protein encoded by the gene Slc10A4 suppresses epileptiform activity and regulates sensitivity to cholinergic chemoconvulsants
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    2013 (English)In: Experimental Neurology, ISSN 0014-4886, E-ISSN 1090-2430, Vol. 239, p. 73-81Article in journal (Refereed) Published
    Abstract [en]

    The expanding number of disease-causing dysfunctions of synaptic proteins illustrates the importance of investigating newly discovered proteins involved in neuronal transmission. The gene Slc10A4 encodes a recently described carrier protein present in pre-synaptic terminals of cholinergic and monoaminergic neurons. The biological significance of this recently described transporter protein is currently unknown. We here investigated whether absence of the Slc10a4 protein has any impact on function of the cholinergic system. We first investigated the sensitivity of Slc10a4 null mice to cholinergic stimulus in vitro. In contrast to wild type mice, gamma oscillations occurred spontaneously in hippocampal slices from Slc10a4 null mice. Furthermore, moderate treatment of Slc10a4 null slices with the cholinergic agonist carbachol induced epileptiform activity. In vivo, 3-channel EEG measurements in freely behaving mice revealed that Slc10a4 null mice had frequent epileptiform spike-activity before treatment, and developed epileptic seizures, detected by EEG and accompanied by observable behavioral components, more rapidly after injection of the cholinergic agonist pilocarpine. Similar results were obtained on non-operated mice, as evaluated by behavioral seizures and post mortem c-Fos immunohistochemistry. Importantly, Slc10a4 null mice and wild type control mice were equally sensitive to the glutamatergic chemoconvulsant kainic acid, demonstrating that absence of Slc10a4 led to a selective cholinergic hypersensitivity. In summary, we report that absence of the recently discovered synaptic vesicle protein Slc10a4 results in increased sensitivity to cholinergic stimulation.

    National Category
    Medical Genetics
    Research subject
    Neurology
    Identifiers
    urn:nbn:se:uu:diva-184841 (URN)10.1016/j.expneurol.2012.09.006 (DOI)000313765000009 ()23022458 (PubMedID)
    Note

    Correction in: EXPERIMENTAL NEUROLOGY  Volume: 247   Pages: 750-750   DOI: 10.1016/j.expneurol.2013.01.030

    Available from: 2012-11-15 Created: 2012-11-15 Last updated: 2018-01-12Bibliographically approved
    3. A role for SLC10A4 in structural remodelling and transmitter release at the mouse neuromuscular junctions
    Open this publication in new window or tab >>A role for SLC10A4 in structural remodelling and transmitter release at the mouse neuromuscular junctions
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    (English)Manuscript (preprint) (Other academic)
    National Category
    Neurosciences
    Identifiers
    urn:nbn:se:uu:diva-214138 (URN)
    Available from: 2014-01-07 Created: 2014-01-07 Last updated: 2018-01-11
  • 78.
    Patra, Kalicharan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Lyons, David J
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Bauer, Pavol
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience.
    Hilscher, Markus M
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Sharma, Swati
    Leão, Richardson N
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Kullander, Klas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    A role for solute carrier family 10 member 4, or vesicular aminergic-associated transporter, in structural remodelling and transmitter release at the mouse neuromuscular junction.2015In: European Journal of Neuroscience, ISSN 0953-816X, E-ISSN 1460-9568, Vol. 41, no 3, p. 316-327Article in journal (Refereed)
    Abstract [en]

    The solute carrier and presynaptic vesicle protein solute carrier family 10 member 4, or vesicular aminergic-associated transporter (VAAT), was recently proven to have a modulatory role in central cholinergic signalling. It is currently unknown whether VAAT also affects peripheral cholinergic synapses. Here we demonstrated a regulatory role for the presynaptic vesicle protein VAAT in neuromuscular junction (NMJ) development and function. NMJs lacking VAAT had fewer branch points, whereas endplates showed an increased number of islands. Whereas the amplitude of spontaneous miniature endplate potentials in VAAT-deficient NMJs was decreased, the amplitude of evoked endplate potentials and the size of the readily releasable pool of vesicles were both increased. Moreover, VAAT-deficient NMJs displayed aberrant short-term synaptic plasticity with enhanced synaptic depression in response to high-frequency stimulation. Finally, the transcript levels of cholinergic receptor subunits in VAAT-deficient muscles were increased, indicating a compensatory postsynaptic sensitization. Our results suggested that VAAT modulates NMJ transmission efficiency and, as such, may represent a novel target for treatment of disorders affecting motor neurons.

  • 79.
    Patra, Kalicharan
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Lyons, David Joseph
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Bauer, Pavol
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Hilscher, Markus M
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Sharma, Swati
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Leao, Richardson N
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Kullander, Klas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    A role for SLC10A4 in structural remodelling and transmitter release at the mouse neuromuscular junctionsManuscript (preprint) (Other academic)
  • 80. Peixoto, Helton M.
    et al.
    Munguba, Hermany
    Cruz, Rossana M. S.
    Guerreiro, Ana M. G.
    Leao, Richardson N.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics.
    Automatic tracking of cells for video microscopy in patch clamp experiments2014In: Biomedical engineering online, ISSN 1475-925X, E-ISSN 1475-925X, Vol. 13, p. 78-Article in journal (Refereed)
    Abstract [en]

    Background: Visualisation of neurons labeled with fluorescent proteins or compounds generally require exposure to intense light for a relatively long period of time, often leading to bleaching of the fluorescent probe and photodamage of the tissue. Here we created a technique to drastically shorten light exposure and improve the targeting of fluorescent labeled cells that is specially useful for patch-clamp recordings. We applied image tracking and mask overlay to reduce the time of fluorescence exposure and minimise mistakes when identifying neurons. Methods: Neurons are first identified according to visual criteria (e.g. fluorescence protein expression, shape, viability etc.) and a transmission microscopy image Differential Interference Contrast (DIC) or Dodt contrast containing the cell used as a reference for the tracking algorithm. A fluorescence image can also be acquired later to be used as a mask (that can be overlaid on the target during live transmission video). As patch-clamp experiments require translating the microscope stage, we used pattern matching to track reference neurons in order to move the fluorescence mask to match the new position of the objective in relation to the sample. For the image processing we used the Open Source Computer Vision (OpenCV) library, including the Speeded-Up Robust Features (SURF) for tracking cells. The dataset of images (n = 720) was analyzed under normal conditions of acquisition and with influence of noise (defocusing and brightness). Results: We validated the method in dissociated neuronal cultures and fresh brain slices expressing Enhanced Yellow Fluorescent Protein (eYFP) or Tandem Dimer Tomato (tdTomato) proteins, which considerably decreased the exposure to fluorescence excitation, thereby minimising photodamage. We also show that the neuron tracking can be used in differential interference contrast or Dodt contrast microscopy. Conclusion: The techniques of digital image processing used in this work are an important addition to the set of microscopy tools used in modern electrophysiology, specially in experiments with neuron cultures and brain slices.

  • 81.
    Perry, Sharn