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Induction of DREADD expression in the rat locus coeruleus and its effects on neuronal firing

(2018)
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Abstract
Aim: Recent research suggests that the Locus Coeruleus-Noradrenaline (LC-NA) system plays a crucial role in vagus nerve stimulation (VNS) effects on brain excitability and seizure control. Selective modulation of the LC with chemogenetics might be a tool to study the role of the LC-NA system in the mechanism of action of VNS. This project will investigate the feasibility to activate LC neurons, expressing the excitatory hM3Dq Designer Receptor Exclusively Activated by Designer Drugs (DREADD), using subclinical doses of clozapine. Methods: Five male Sprague-Dawley rats were injected bilaterally with 10nl of adeno-associated viral vector AAV2/7-PRSx8-hM3Dq-mCherry (n=2) or AAV2/7-PRSx8-eGFP (n=3) in the LC. After three weeks, tungsten microelectrodes were positioned in the LC of isoflurane-anesthetized rats to record action potentials. After 300s baseline recording, clozapine was injected in increasing doses (0.01mg/kg, 0.1mg/kg; s.c.). Subsequently, a decreased firing rate after clonidine (0.04mg/kg, s.c.) injection was used to confirm LC identity. Average firing frequency between 300 and 900s after clozapine injections were compared to 300s baseline. For determination of hM3Dq-mcherry expression levels, immunofluorescence staining was performed. Results: Recordings were obtained from five neurons in hM3Dq-animals and three in eGFP-animals. While 0.01mg/kg clozapine had no effect on firing frequency of eGFP-neurons (n=3), 0.1mg/kg clozapine increased firing frequency to 1.50±0.43Hz compared with baseline (1.17±0.39Hz). In four hM3Dq-neurons, 0.01mg/kg clozapine increases firing rate to 5.28±2.64Hz, 0.1mg/kg to 5.86±2.93Hz compared with baseline (4.08±1.22Hz). One hM3Dq-neuron was characterized by decreases in firing rate after clozapine (0.01mg/kg:1.56 Hz; 0.1mg/kg:1.37Hz) compared to baseline (2.00 Hz). Immunofluorescence staining revealed good expression in dorsal LC. Conclusions: At the lowest dose tested (0.01mg/kg) clozapine did not influence LC firing frequencies in eGFP-neurons, while it increased or decreased firing frequencies in hM3Dq-neurons. Decreased firing could indicate that recorded neurons did not express DREADDs and were subjected to hetero-inhibition induced by NA-release from surrounding DREADD-expressing neurons.
Keywords
chemogenetics, locus coeruleus, vagus nerve stimulation

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Chicago
Stevens, Latoya, Robrecht Raedt, Wouter Van Lysebettens, Veerle Baekelandt, Chris Van den Haute, Paul Boon, and Kristl Vonck. 2018. “Induction of DREADD Expression in the Rat Locus Coeruleus and Its Effects on Neuronal Firing.” In .
APA
Stevens, Latoya, Raedt, R., Van Lysebettens, W., Baekelandt, V., Van den Haute, C., Boon, P., & Vonck, K. (2018). Induction of DREADD expression in the rat locus coeruleus and its effects on neuronal firing. Presented at the Research day and student symposium .
Vancouver
1.
Stevens L, Raedt R, Van Lysebettens W, Baekelandt V, Van den Haute C, Boon P, et al. Induction of DREADD expression in the rat locus coeruleus and its effects on neuronal firing. 2018.
MLA
Stevens, Latoya, Robrecht Raedt, Wouter Van Lysebettens, et al. “Induction of DREADD Expression in the Rat Locus Coeruleus and Its Effects on Neuronal Firing.” 2018. Print.
@inproceedings{8576790,
  abstract     = {Aim: Recent research suggests that the Locus Coeruleus-Noradrenaline (LC-NA) system plays a crucial role in vagus nerve stimulation (VNS) effects on brain excitability and seizure control. Selective modulation of the LC with chemogenetics might be a tool to study the role of the LC-NA system in the mechanism of action of VNS. This project will investigate the feasibility to activate LC neurons, expressing the excitatory hM3Dq Designer Receptor Exclusively Activated by Designer Drugs (DREADD), using subclinical doses of clozapine.
Methods: Five male Sprague-Dawley rats were injected bilaterally with 10nl of adeno-associated viral vector AAV2/7-PRSx8-hM3Dq-mCherry (n=2) or AAV2/7-PRSx8-eGFP (n=3) in the LC. After three weeks, tungsten microelectrodes were positioned in the LC of isoflurane-anesthetized rats to record action potentials. After 300s baseline recording, clozapine was injected in increasing doses (0.01mg/kg, 0.1mg/kg; s.c.). Subsequently, a decreased firing rate after clonidine (0.04mg/kg, s.c.) injection was used to confirm LC identity. Average firing frequency between 300 and 900s after clozapine injections were compared to 300s baseline. For determination of hM3Dq-mcherry expression levels, immunofluorescence staining was performed.
Results: Recordings were obtained from five neurons in hM3Dq-animals and three in eGFP-animals. While 0.01mg/kg clozapine had no effect on firing frequency of eGFP-neurons (n=3), 0.1mg/kg clozapine increased firing frequency to 1.50{\textpm}0.43Hz compared with baseline (1.17{\textpm}0.39Hz). In four hM3Dq-neurons, 0.01mg/kg clozapine increases firing rate to 5.28{\textpm}2.64Hz, 0.1mg/kg to 5.86{\textpm}2.93Hz compared with baseline (4.08{\textpm}1.22Hz). One hM3Dq-neuron was characterized by decreases in firing rate after clozapine (0.01mg/kg:1.56 Hz; 0.1mg/kg:1.37Hz) compared to baseline (2.00 Hz). Immunofluorescence staining revealed good expression in dorsal LC.
Conclusions: At the lowest dose tested (0.01mg/kg) clozapine did not influence LC firing frequencies in eGFP-neurons, while it increased or decreased firing frequencies in hM3Dq-neurons. Decreased firing could indicate that recorded neurons did not express DREADDs and were subjected to hetero-inhibition induced by NA-release from surrounding DREADD-expressing neurons.  

},
  author       = {Stevens, Latoya and Raedt, Robrecht and Van Lysebettens, Wouter and Baekelandt, Veerle  and Van den Haute, Chris  and Boon, Paul and Vonck, Kristl},
  keyword      = {chemogenetics,locus coeruleus,vagus nerve stimulation},
  location     = {Ghent University},
  title        = {Induction of DREADD expression in the rat locus coeruleus and its effects on neuronal firing},
  year         = {2018},
}