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Functional Aspects of Peripheral and Spinal Cord Neurons Involved in Itch and Pain
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Neuroscience, Developmental Genetics. (Sensory Circuits)ORCID iD: 0000-0002-1386-1307
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 [en]
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: urn:nbn:se:uu:diva-284070ISBN: 978-91-554-9568-8 (print)OAI: oai:DiVA.org:uu-284070DiVA: diva2:919731
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
List of papers
1. Identification of a Neuronal Receptor Controlling Anaphylaxis
Open this publication in new window or tab >>Identification of a Neuronal Receptor Controlling Anaphylaxis
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2016 (English)In: Cell reports, ISSN 2211-1247, E-ISSN 2211-1247, Vol. 14, no 2, 370-379 p.Article in journal (Refereed) Published
Abstract [en]

Allergic reactions can in severe cases induce a state of circulatory shock referred to as anaphylaxis. Histamine, the primary mediator of this condition, is released from immune cells, and, therefore, anaphylaxis has so far been considered an immune system disorder. However, we here show that the glutamatergic receptor mGluR7, expressed on a subpopulation of both peripheral and spinal cord neurons, controls histamine-induced communication through calcium-dependent autoinhibition with implications for anaphylaxis. Genetic ablation of mGluR7, and thus altered regulation of histamine-sensing neurons, caused an anaphylaxis-like state in mGluR7(-/-) mice, which could be reversed by antagonizing signaling between neurons and mast cells but not by antagonizing a central itch pathway. Our findings demonstrate the vital role of nervous system control by mGluR7 in anaphylaxis and open up possibilities for preventive strategies for this life-threatening condition.

National Category
Neurosciences
Identifiers
urn:nbn:se:uu:diva-272637 (URN)10.1016/j.celrep.2015.12.033 (DOI)000368101600019 ()26748715 (PubMedID)
Funder
Swedish Research CouncilRagnar Söderbergs stiftelseKnut and Alice Wallenberg FoundationÅke Wiberg FoundationMagnus Bergvall FoundationThe Royal Swedish Academy of Sciences
Available from: 2016-01-15 Created: 2016-01-15 Last updated: 2017-11-30Bibliographically approved
2. Spinal Cord Interneurons Expressing the Gastrin-Releasing Peptide Receptor Convey Itch Through VGLUT2-Mediated Signaling
Open this publication in new window or tab >>Spinal Cord Interneurons Expressing the Gastrin-Releasing Peptide Receptor Convey Itch Through VGLUT2-Mediated Signaling
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2017 (English)In: Pain, ISSN 0304-3959, E-ISSN 1872-6623, Vol. 158, no 5, 945-961 p.Article in journal (Refereed) Published
Abstract [en]

Itch is a sensation that promotes the desire to scratch, which can be evoked by mechanical and chemical stimuli. In the spinal cord, neurons expressing the gastrin-releasing peptide receptor (GRPR) have been identified as specific mediators of itch. However, our understanding of the GRPR population in the spinal cord, and thus how these neurons exercise their functions, is limited. For this purpose, we constructed a Cre line designed to target the GRPR population of neurons (Grpr-Cre). Our analysis revealed that Grpr-Cre cells in the spinal cord are predominantly excitatory interneurons that are found in the dorsal lamina, especially in laminae II-IV. Application of the specific agonist gastrin-releasing peptide induced spike responses in 43.3% of the patched Grpr-Cre neurons, where the majority of the cells displayed a tonic firing property. Additionally, our analysis showed that the Grpr-Cre population expresses Vglut2 mRNA, and mice ablated of Vglut2 in Grpr-Cre cells (Vglut2-lox; Grpr-Cre mice) displayed less spontaneous itch and attenuated responses to both histaminergic and nonhistaminergic agents. We could also show that application of the itch-inducing peptide, natriuretic polypeptide B, induces calcium influx in a subpopulation of Grpr-Cre neurons. To summarize, our data indicate that the Grpr-Cre spinal cord neural population is composed of interneurons that use VGLUT2-mediated signaling for transmitting chemical and spontaneous itch stimuli to the next, currently unknown, neurons in the labeled line of itch.

Keyword
Itch, Gastrin-releasing peptide receptor population, Natriuretic polypeptide B, Spinal cord, Vesicular glutamate transporter 2, Neuronal networks, Labeled line of itch, Electrophysiology, Conditional knockout analysis, Tracing, Calcium imaging, Grpr, VGLUT2
National Category
Medical and Health Sciences
Research subject
Medical Science
Identifiers
urn:nbn:se:uu:diva-284058 (URN)10.1097/j.pain.0000000000000861 (DOI)000402430600021 ()28157737 (PubMedID)
Funder
Swedish Research CouncilThe Swedish Brain FoundationThe Royal Swedish Academy of SciencesRagnar Söderbergs stiftelseMagnus Bergvall FoundationGunvor och Josef Anérs stiftelse
Note

Title in thesis list of papers: Spinal Cord Interneurons Expressing the Gastrin Releasing Peptide Receptor Convey Itch through VGLUT2-mediated Signaling

Available from: 2016-04-14 Created: 2016-04-14 Last updated: 2017-07-10Bibliographically approved
3. Characterization of preprodynorphin-expressing cells in the mouse nervous system
Open this publication in new window or tab >>Characterization of preprodynorphin-expressing cells in the mouse nervous system
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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
Preprodynorphin, characterization
National Category
Medical and Health Sciences
Research subject
Medical Science
Identifiers
urn:nbn:se:uu:diva-284066 (URN)
Available from: 2016-04-14 Created: 2016-04-14 Last updated: 2016-06-01Bibliographically approved
4. Characterization of preproenkephalin expressing neurons in the nervous system using a transgenic line
Open this publication in new window or tab >>Characterization of preproenkephalin expressing neurons in the nervous system using a transgenic line
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Here we have constructed a Cre line to target preproenkephalin expressing cells (Penk-Cre) using a BAC cloning strategy. By crossing our Penk-Cre line with the tdTomato reporter, tdTomato;Penk-Cre expressing cells could be visualized. Penk-Cre was expressed throughout the dorsal spinal cord, where approximately 50% of the neurons were residing within lamina III. Furthermore, single-cell analysis of spinal Penk-Cre expressing cells showed that 41% was positive for Penk mRNA and that a majority (94%) of the population expressed Vglut2 mRNA. Immunohistochemical analysis showed that only 7% and 13% expressed the inhibitory markers Viaat-egfp and Pax2, respectively, hence identifying spinal Penk-Cre expressing neurons as mainly excitatory. The expression of Penk-Cre in the brain was extensive, including dense expression in the striatum, nucleus accumbens and several amygdaloid nuclei. Furthermore, Penk-Cre expression was also shown in insular cortex, cingulated cortex, primary and secondary somatosensory cortices and the trigeminal nuclei. The Penk-Cre line represents a useful genetic tool for future analysis of PENK expressing neurons in the nervous system.

 

Keyword
characterization, Cre, preproenkephalin, Penk
National Category
Medical and Health Sciences
Research subject
Medical Science
Identifiers
urn:nbn:se:uu:diva-284068 (URN)
Available from: 2016-04-14 Created: 2016-04-14 Last updated: 2016-06-01Bibliographically approved

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