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

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Abstract
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.

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MLA
Stevens, Latoya, et al. “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, 2019, doi:10.3389/conf.fnins.2019.96.00009.
APA
Stevens, L., Vonck, K., Van Lysebettens, W., Baekelandt, V., Van Den Haute, C., Carrette, E., … Raedt, R. (2019). Genetic modification of Locus Coeruleus NE cells for chemogenetic activation remains challenging. In Front. Neurosci. Conference Abstract: 13th National Congress of the Belgian Society for Neuroscience. Brussels. https://doi.org/10.3389/conf.fnins.2019.96.00009
Chicago author-date
Stevens, Latoya, Kristl Vonck, Wouter Van Lysebettens, Veerle Baekelandt, Chris Van Den Haute, Evelien Carrette, Paul Boon, and Robrecht Raedt. 2019. “Genetic Modification of Locus Coeruleus NE Cells for Chemogenetic Activation Remains Challenging.” In Front. Neurosci. Conference Abstract: 13th National Congress of the Belgian Society for Neuroscience. https://doi.org/10.3389/conf.fnins.2019.96.00009.
Chicago author-date (all authors)
Stevens, Latoya, Kristl Vonck, Wouter Van Lysebettens, Veerle Baekelandt, Chris Van Den Haute, Evelien Carrette, Paul Boon, and Robrecht Raedt. 2019. “Genetic Modification of Locus Coeruleus NE Cells for Chemogenetic Activation Remains Challenging.” In Front. Neurosci. Conference Abstract: 13th National Congress of the Belgian Society for Neuroscience. doi:10.3389/conf.fnins.2019.96.00009.
Vancouver
1.
Stevens L, Vonck K, Van Lysebettens W, Baekelandt V, Van Den Haute C, Carrette E, et al. Genetic modification of Locus Coeruleus NE cells for chemogenetic activation remains challenging. In: Front Neurosci Conference Abstract: 13th National Congress of the Belgian Society for Neuroscience. 2019.
IEEE
[1]
L. Stevens et al., “Genetic modification of Locus Coeruleus NE cells for chemogenetic activation remains challenging,” in Front. Neurosci. Conference Abstract: 13th National Congress of the Belgian Society for Neuroscience, Brussels, 2019.
@inproceedings{8653408,
  abstract     = {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.},
  author       = {Stevens, Latoya and Vonck, Kristl and Van Lysebettens, Wouter and Baekelandt, Veerle and Van Den Haute, Chris and Carrette, Evelien and Boon, Paul and Raedt, Robrecht},
  booktitle    = {Front. Neurosci. Conference Abstract: 13th National Congress of the Belgian Society for Neuroscience},
  language     = {eng},
  location     = {Brussels},
  title        = {Genetic modification of Locus Coeruleus NE cells for chemogenetic activation remains challenging},
  url          = {http://dx.doi.org/10.3389/conf.fnins.2019.96.00009},
  year         = {2019},
}

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