Genetic modification of Locus Coeruleus NE cells for chemogenetic activation remains challenging

Event Abstract Back to Event Genetic modification of Locus Coeruleus NE cells for chemogenetic activation remains challenging Latoya Stevens1*, Kristl Vonck1, Wouter Van Lysebettens1, Veerle Baekelandt2, Chris Van Den Haute3, Evelien Carrette1, Paul Boon1 and Robrecht Raedt1 1 4Brain, Department of Head and Skin, Ghent University, Belgium 2 Laboratory for Neurobiology and Gene therapy, Center for Molecular Medicine and Leuven Brain Institute, Belgium 3 Leuven Viral Vector Core, Centre for Molecular Medicine, Belgium Aim: The Locus Coeruleus-Noradrenaline (LC-NE) system plays an important role in vagus nerve stimulation (VNS) induced effects on brain excitability and epileptic seizure control. Chemogenetic approaches to selectively modulate the LC may allow dedicated investigation of the role of the LC-NE pathway in the regulation of brain excitability. This study investigated the feasibility to express the excitatory hM3Dq DREADD (Designer Receptor Exclusively Activated by Designer Drugs) in LC neurons and perform unit recording of genetically modified LC neurons. Methods: Thirty-seven male Sprague-Dawley rats were injected with 10nl of adeno-associated viral vector AAV2/7-PRSx8-hM3Dq-mCherry (n=20) or AAV2/7-PRSx8-eGFP (n=17) in the LC (3.9AP; 1.15ML; -5.7DV) using a Neuro-Syringe (Hamilton 7001 point style 3). Three weeks later LC unit recordings were performed in anesthetized rats to investigate the effects of clozapine (0.01 and 0.1 mg/kg) as DREADD ligand to activate the modified LC neurons. After characterization of LC-NE neuron by the occurrence of ‘a phasic burst inhibition’ after a foot pinch, baseline activity was recorded followed by subsequent systemic administration of 0.01 mg/kg and 0.1 mg/kg clozapine. Subsequently, a decreased firing rate after clonidine (0.04 mg/kg, s.c.) injection was used to confirm LC identity of the recorded neuron. Animals were euthanized at the end of the electrophysiological recordings for determination of hM3Dq-mcherry expression levels using immunofluorescence staining. Results: Successful electrophysiological recordings were performed in 12 animals. A total of 12 neurons were recorded, 5 in control animals and 7 in DREADD-injected animals, all responding with a decreased firing frequency after clonidine administration. There was no significant difference in pre-clozapine mean baseline LC firing frequency between DREADD (2.67 ± 0.26 Hz) and control LC neurons (1.88 ± 0.61 Hz; P=0.21, unpaired t test). Systemic administration of the lowest dose of clozapine (0.01 mg/kg) had no clear effect on the mean firing frequency of recorded LC neurons whereas an increased firing rate was observed after the highest dose (0.1 mg/kg) irrespective whether they were recorded from DREADD or control animals (P=0.006, two way RM ANOVA). Co-labeling of LC neurons (via expression of dopamine-β-hydroxylase, DBH) and mCherry-tag showed that 21 ± 2.3% LC neurons are expressing the hM3Dq receptor (21 sections; three sections/animal; range 11-32%). Aspecific expression of hM3Dq-mCherry was also observed in DBH- cells (26 ± 4.1%). Conclusion: LC unit recording is feasible following manipulations for DREADD induction. In the current experimental set-up we were not able to draw conclusions on the feasibility for chemogenetic modulation of LC neurons with clozapine due to a low efficiency and selectivity of LC neuron transduction. At doses of 0.1 mg/kg clozapine increases the firing rate of LC neurons irrespective of whether they express hM3Dq. Acknowledgements Latoya Stevens is supported by a PhD-grant from the Queen Elisabeth Medical Foundation for Neurosciences (G.S.K.E) and the Ghent Institute for Neuroscience. Keywords: Locus coeruleus (LC), DREADD = designer receptor exclusively activated by designer drugs, Unit recordings, Clozapine, Epilepsy Conference: 13th National Congress of the Belgian Society for Neuroscience , Brussels, Belgium, 24 May - 24 May, 2019. Presentation Type: Poster presentation Topic: Behavioral/Systems Neuroscience Citation: Stevens L, Vonck K, Van Lysebettens W, Baekelandt V, Van Den Haute C, Carrette E, Boon P and Raedt R (2019). Genetic modification of Locus Coeruleus NE cells for chemogenetic activation remains challenging. Front. Neurosci. Conference Abstract: 13th National Congress of the Belgian Society for Neuroscience . doi: 10.3389/conf.fnins.2019.96.00009 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 30 Apr 2019; Published Online: 27 Sep 2019. * Correspondence: Ms. Latoya Stevens, 4Brain, Department of Head and Skin, Ghent University, Ghent, Belgium, latoya.stevens@ugent.be Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Latoya Stevens Kristl Vonck Wouter Van Lysebettens Veerle Baekelandt Chris Van Den Haute Evelien Carrette Paul Boon Robrecht Raedt Google Latoya Stevens Kristl Vonck Wouter Van Lysebettens Veerle Baekelandt Chris Van Den Haute Evelien Carrette Paul Boon Robrecht Raedt Google Scholar Latoya Stevens Kristl Vonck Wouter Van Lysebettens Veerle Baekelandt Chris Van Den Haute Evelien Carrette Paul Boon Robrecht Raedt PubMed Latoya Stevens Kristl Vonck Wouter Van Lysebettens Veerle Baekelandt Chris Van Den Haute Evelien Carrette Paul Boon Robrecht Raedt Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.