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Characterization of preprodynorphin-expressing cells in the mouse nervous system
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics. (Sensory Circuits)ORCID iD: 0000-0002-1386-1307
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics. (Sensory Circuits)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics. (Sensory Circuits)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics. (Sensory Circuits)
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Dynorphin is an endogenous opioid that has been implicated in maintaining chronic pain and gating of both itch and acute mechanical pain through acting on both opioid and non-opioid receptors. To improve our understanding of the complex and multifunctional population that expresses dynorphin, we have constructed a preprodynorphin Cre line (Pdyn-Cre) using BAC cloning. Single cell analysis of tdTomato;Pdyn-Cre cells revealed that 43% of the population expressed Pdyn mRNA, and no analyzed tdTomato;Pdyn-Cre negative cell expressed Pdyn mRNA, thus confirming that Cre had been inserted under the control of the Pdyn promoter. The Pdyn-Cre expressing population was found in the dorsal spinal cord, mainly in lamina II-IV and overlapped to 47% with Vglut2 mRNA, while co-expression with the inhibitory markers Viaat-egfp and Pax2 was 13% and 28%, respectively. The expression of Pdyn-Cre in the brain was extensive, marking virtually all cortical structures, including somatosensory and motor cortex. Furthermore, Pdyn-Cre was densely expressed in the striatum, amygdala and parts of the hippocampus, and expression was also observed in several pain and itch processing areas, including amygdala, lateral parabrachial nucleus, claustrum, insular cortex and raphe magnus nucleus. Our analysis indicates that the transgenic Pdyn-Cre line includes PDYN cells in the nervous system and will thus be useful as a transgenic tool for studies of the role and connectivity of the PDYN population.

Keyword [en]
Preprodynorphin, characterization
National Category
Medical and Health Sciences
Research subject
Medical Science
Identifiers
URN: urn:nbn:se:uu:diva-284066OAI: oai:DiVA.org:uu-284066DiVA: diva2:919702
Available from: 2016-04-14 Created: 2016-04-14 Last updated: 2016-06-01Bibliographically approved
In thesis
1. Functional Aspects of Peripheral and Spinal Cord Neurons Involved in Itch and Pain
Open this publication in new window or tab >>Functional Aspects of Peripheral and Spinal Cord Neurons Involved in Itch and Pain
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

We have investigated the role of the metabotropic glutamate receptor 7 (mGluR7) and the gastrin releasing peptide receptor (Grpr) population that are involved at different levels of itch transmission. We found that mGuR7 deficient mice displayed an anaphylaxis-like behavior when provoked with histamine. Analysis of blood revealed elevated plasma levels of histamine and mouse mast cell protease-1 (mMCP1), two indicators of anaphylaxis, in mGluR7 deficient mice compared with control mice. Inhibition of the neurokinin 1 receptor, by preventing binding of the corresponding ligand substance P (SP), prior to provocation with histamine prevented the development of anaphylaxis in mGluR7 deficient animals. However, blocking GRPR (gastrin releasing peptide receptor) only resulted in decreased itch levels in mGluR7 deficient mice but did not prevent the systemic anaphylaxis-like behavior. Our findings indicate that mGluR7 normally functions as a brake on histaminergic itch that is mediated through GRPR as well as anaphylaxis through Substance P.

Grpr has previously been shown to mediate both histaminergic and non-histaminergic itch but little is known about the GRPR neuronal population. We used a BAC cloning strategy to construct a Grpr-Cre line, which we crossed with the reporter lines tdTomato and Viaat-egfp as well as with Vglut2-lox. We could conclude that Grpr-Cre neurons are mainly excitatory interneurons located in lamina II-IV, that convey itch using VGLUT2-mediated glutamatergic transmission to the next, currently unknown, step in the labeled line of chemical itch.

To eventually deduce the function of the endogenous opioids dynorphin and enkephalin, which are hypothesized to be involved in gating pain and itch in the spinal cord, we constructed two Cre lines using BAC cloning that targeted the precursor proteins preprodynorphin and preproenkephalin, respectively. Preprodynorphin-Cre neurons were mainly located in lamina II-IV and overlapped to 47% with Vglut2 mRNA, while the co-expression with the inhibitory markers Viaat-egfp and PAX2 was 13% and 28% respectively in the spinal cord. Preproenkephalin neurons were more localized to lamina III in the dorsal horn, furthermore single cell analysis showed that they overlapped to 94% with Vglut2 mRNA while 7% and 13% expressed Viaat-egfp and PAX2 respectively.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. 48 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 1222
Keyword
mGluR7, anaphylaxis, Grpr, Penk, Pdyn, Cre line, BAC cloning, spinal cord, transgenic line
National Category
Medical and Health Sciences
Research subject
Medical Science
Identifiers
urn:nbn:se:uu:diva-284070 (URN)978-91-554-9568-8 (ISBN)
Public defence
2016-06-04, C2:301, Husargatan 3, Uppsala, 09:00 (English)
Opponent
Supervisors
Available from: 2016-05-13 Created: 2016-04-14 Last updated: 2016-06-01

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