0148 Serotonergic Dorsal Raphe Neurons Regulate Hypercapnia Induced Arousal Through 5HT2A Receptors on the Parabrachial Neurons

Introduction Serotoninergic dorsal raphe neurons (DRSert) are CO2 responsive, and mice lacking serotonin have impaired arousal to CO2. We showed that the neurons in external lateral parabrachial nucleus containing calcitonin gene related peptide (PBelCGRP), are required for CO2-arousal. PBelCGRP neurons also receive serotoninergic innervation from the DRSert. 5HT2A agonist restores CO2 responsiveness in mice lacking serotonin, suggesting that DRSert may modulate CO2 arousal by acting on 5HT2A receptors possibly on the PBel neurons. Methods We used serotonin transporter (Sert)-Cre mice to optogenetically inhibit DRSert neurons and their terminals in the PBel. We injected AAV-FLEX-ArchT into the DR and implanted an optical fiber just above it in one set of Sert-Cre mice and bilaterally in the PBel in another set. All mice were instrumented for sleep and optogenetics and were tested for EEG arousals to 10% CO2. Latencies of arousal were compared with optogenetic inhibition of either the DR neurons or their terminals in the PBel with a 593nm laser light. We further tested whether a 5HT2A agonist (TCB-2) can reverse blockade of CO2 arousal in mice where DRSert terminals in PBel were inhibited. Finally, TCB-2 was injected in mice with PBelCGRP deletions and arousal latency to CO2 was compared. Results Compared to the control (Laser-OFF) condition, arousal latency to CO2 was significantly increased by photoinhibition of either the DRSert neurons (n=6; latency- 40.9 ± 6.4 vs. 13.81± 0.69 sec; F3, 17= 11.5; P< 0.001) or their terminals in PBel (n=8; latency-34.9 ± 2.3 sec vs. 16.62 ± 0.97sec, F1, 14= 56.9; P< 0.001). This was reversed by the 5HT2A agonist TCB-2 (5mg/kg), as it reduced the latency to CO2 arousal in mice with photoinhibition of terminals in PBel from 35.48 ± 7.31 sec to 16.24 ± 1.06 sec (F3, 9= 8.05; P= 0.006), but had no effect in mice with PBelCGRP neurons deletions. Conclusion The serotonin system modulate CO2-arousals by the DRSert input to the PBel. TCB-2 reversed the effect of inhibition of DRSert terminals in the PBel, but not in mice with PBelCGRP deletions, suggests that DRSert modulate PBelCGRP neurons through 5HT2a receptors. Support NIH- 2P01 HL095491 and NS112175

Classical neurotransmitters in the ovijector of Ascaris suum: localization and modulation of muscle activity

Ascaris suum possesses a well-developed nervous system which is regulated by a number of classical neurotransmitters including acetylcholine (ACh), γ-aminobutyric acid (GABA), glutamate and serotonin. The vagina vera, the distal part of the ovijector, displays intrinsic, rhythmic activity which has been shown to be modulated by FMRFamide-related peptides (FaRPs) in vitro. Confocal scanning laser microscopy coupled with immunocytochemistry, and histochemical studies, revealed that the nerve plexus of the ovijector contains GABAergic and glutamatergic innervation. Although no distinctive cholinergic or serotoninergic innervation was apparent, cholinesterase activity was localized to discrete areas of the musculature of the vagina vera. The effects of classical transmitters on the activity of the vagina vera in vitro were examined. ACh was excitatory, stimulating a brief but powerful contraction of the vagina vera with a threshold for activity of 1 μM. Both GABA and glutamate were inhibitory, causing a cessation of contractile activity at high concentrations (> 10 μM). Although less potent than glutamate, GABA had more profound effects and induced longer-lasting paralysis of the tissue. The threshold concentrations for activity were 5 μM for glutamate and 10 μM for GABA. Serotonin had no consistent effect on the vagina vera. This study demonstrates that classical transmitters modulate the activity of the ovijector of A. suum.