A Feasibility Study to Investigate Chemogenetic Modulation of the Locus Coeruleus by Means of Single Unit Activity

Abstract

Aim: Selective chemogenetic modulation of locus coeruleus (LC) neurons would allow dedicated investigation of the role of the LC-NA pathway in brain excitability and disorders such as epilepsy. This study investigated the feasibility of an experimental set-up where chemogenetic modification of the brainstem locus coeruleus NA neurons is aimed at and followed by LC unit activity recording in response to clozapine.Methods: The LC of male Sprague-Dawley rats was injected with 10 nl of adenoassociated viral vector AAV2/7-PRSx8-hM3Dq-mCherry (n = 19, DREADD group) or AAV2/7-PRSx8-eGFP (n = 13, Controls). Three weeks later, LC unit recordings were performed in anesthetized rats. We investigated whether clozapine, a drug known to bind to modified neurons expressing hM3Dq receptors, was able to increase the LC firing rate. Baseline unit activity was recorded followed by subsequent administration of 0.01 and 0.1 mg/kg of clozapine in all rats. hM3Dq-mcherry expression levels were investigated using immunofluorescence staining of brainstem slices at the end of the experiment.Results: Unit recordings could be performed in 12 rats and in a total of 12 neurons (DREADDs: n = 7, controls: n = 5). Clozapine 0.01 mg/kg did not affect the mean firing rate of recorded LC-neurons; 0.1 mg/kg induced an increased firing rate, irrespective whether neurons were recorded from DREADD or control rats (p = 0.006). Co-labeling of LC neurons and mCherry-tag showed that 20.6 ± 2.3% LC neurons expressed the hM3Dq receptor. Aspecific expression of hM3Dq-mCherry was also observed in non-LC neurons (26.0 ± 4.1%).Conclusion: LC unit recording is feasible in an experimental set-up following manipulations for DREADD induction. A relatively low transduction efficiency of the used AAV was found. In view of this finding, the effect of injected clozapine on LC-NA could Frontiers in Neuroscience | www.frontiersin.org 1 March 2020 | Volume 14 | Article 162 Stevens et al. Chemogenetics to Investigate LC-NA Pathwaynot be investigated as a reliable outcome parameter for activation of chemogenetically modified LC neurons. The use of AAV2/7, a vector previously applied successfully to target dopaminergic neurons in the substantia nigra, leads to insufficient chemogenetic modification of the LC compared to transduction with AAV2/9.

Optimized Parameters for Transducing the Locus Coeruleus Using Canine Adenovirus Type 2 (CAV2) Vector in Rats for Chemogenetic Modulation Research

“…Transduction levels are the highest when diluted conditions, 0.1E9 pp and 1E9 pp of CAV2-PRSx8-hM3Dq-mCherry at the target site, are used with average expression ranging from 67.9 ± 8.3 to 87.3 ± 9.8%. This is much higher compared to our previous AAV2/7 approach (20.6 ± 2.3%; 5.99 × 10 9 GC/µl) (Stevens et al, 2020) and similar to other groups using the CAV2 viral vector to induce expression of Channelrhodopsin under control of a PRSx8 promotor at conditions similar to ours (3 injections of 400nl, a total of 10 9 viral particles at the target site) observing expression levels of 83 ± 3.4% (Hayat et al, 2020). Low expression levels of hM3Dq were observed with the highest numbers of viral particles (>1E9 pp).…”

“…Viral vectors (both AAV and CAV2) containing PRSx8 driven plasmids are widely used and validated to induce selective transduction in noradrenergic neurons both in mice and rats (Vazey and Aston-Jones, 2014;Fortress et al, 2015;Li et al, 2016;Hirschberg et al, 2017;Kane et al, 2017;Rorabaugh et al, 2017;Vazey et al, 2018;Cope et al, 2019;Xiang et al, 2019;Hayat et al, 2020). In a previous study, using AAV2/7 viral vector to transduce cells with the PRSx8-hMD3q-mCherry construct, we observed low levels of hM3Dq expression in LC (20.6 ± 2.3%) with high levels of aspecific expression (26.0 ± 4.1%) outside the LC (Stevens et al, 2020). These observations indicate that CAV2 is a more efficient tool than AAV2/7 to introduce genes in LC neurons and that PRSx8 can result in important leakage of expression in non-catecholaminergic neurons.…”

“…Our previous results demonstrated low cell-specific expression and specificity using the AAV2/7-PRSx8-hM3Dq-mCherry. We hypothesized that this is due to low tropism of the AAV2/7 viral particle for LC noradrenergic cells (Stevens et al, 2020). The CAV2 viral vector has been proven to efficiently transduce neurons by uptake both at the cell body and at the axonal terminals (Kremer et al, 2000;Soudais et al, 2000;Junyent and Kremer, 2015;Del Rio et al, 2019) and is validated to target the LC (Li et al, 2016;Hirschberg et al, 2017;Hayat et al, 2020).…”

Level of hM4D(Gi) DREADD Expression Determines Inhibitory and Neurotoxic Effects in the Hippocampus

“…There are some reports on neuronal loss and degeneration of glia induced by AAV-mediated overexpression of other proteins in in vivo studies, that did not occur in control conditions where green fluorescent protein was expressed ( Golebiowski et al, 2017 ; Amado et al, 2019 ). One study reports toxicity when hM3(Dq) is expressed in the locus coeruleus of rats following CAV-mediated transduction ( Stevens et al, 2020 ). Neurotoxicity, however, has been reported with green fluorescent protein expression too when high titers were used, even with subretinal administration of AAV, which has applications that are clinically approved ( Howard et al, 2008 ; Samaranch et al, 2014 ; Khabou et al, 2018 ; Xiong et al, 2019 ).…”

“…In contrast to AAV vectors, which are generally considered safe and are currently being tested in several Phase I and II clinical trials, not much is known on the safety profile and recommended viral doses of the DREADD technology itself ( Wang et al, 2019 ). The viral titers and injected volumes reported in literature on preclinical DREADD research in rodents are highly variable, ranging from E + 9 to E + 13 vector genomes (vg)/ml and 0.01–5 μl per injection site, respectively ( Wirtshafter and Stratford, 2016 ; Rorabaugh et al, 2017 ; Wunsch et al, 2017 ; Maharjan et al, 2018 ; Panoz-Brown et al, 2018 ; Dygalo et al, 2020 ; Stevens et al, 2020 ). The minimal DREADD expression level (determined by the AAV serotype and titer, the promoter sequence and the injected volume) required to result in effective neuromodulation is unknown.…”