The role of 5HT1a and 5HT2a receptors within the preBötzinger complex (pBC) and ventral respiratory column (VRC) in eliciting respiratory response to systemic administration of selective serotonin agonists

Abstract

Nedavna istraživanja pokazala su kako selektivni agonisti 5HT receptora mogu spriječiti opioidima induciranu depresiju disanja bez djelovanja na njihov analgetski učinak. Primijenjeni sistemski (intravenski tj. IV) imaju modulirajući učinak na disanje, ali je mjesto djelovanja nepoznato. PreBötzinger kompleks (pBC) smatra se mogućim mjestom. Ova studija napravljena je u svrhu određivanja uloge 5HT1A i 5HT2A receptora unutar pBC kompleksa i ventalne respiratorne grupe (VRC) pri promjeni ritma disanja, a koja nastaje nakon sistemske primjene agonista istih receptora. Korišteni su agonist 5HT1A receptora 8OH-DPAT ((±)-8-Hydroxy-2- dipropylaminotetralin hydrobromide) i agonist 5HT2A receptora DOI ((±)-1-(2,5- Dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride). Pokusi su provođeni na vagotomiziranim, decerebriranim, paraliziranim i mehanički ventiliranim psima. Primijenjena je inhalacijska anestezija maskom i održavana isofluranom (ISO) (1.5- 2,5% end-tidal koncentracija (1-2 MAC)). Istodobno su praćeni znakovi nedovoljne anestezije kao što su salivacija, lakrimacija, porast krvnog tlaka i pulsa, te je po potrebi anestezija produbljivana. Zatim je napravljena decerebracija midkolikularnom transekcijom. Nakon decerebracije prekinut je isofluran (kako bi se otklonio moguć utjecaj anestezije na rezultate), a ventilacija nastavljena mješavinom ~60% zrak i O2 te je održavana hiperoksična normokapnija (Fi > 0,6). Staklena elektroda korištena je za pronalazak i snimanje neuralne aktivnosti respiratornih neurona unutar pBC kompleksa i VRC te za aplikaciju testiranih supstanci. Testirane supstance administrirane su i sistemski (IV). Tijekom pokusa kontinuirano je praćen neurogram freničnog živca (PNG), frekvencija izbijanja respiratornih neurona, vrijeme inspirija (TI) i ekspirija (TE), etCO2 te sistemski krvni tlak. Za lokalizaciju pBC kompleksa unutar ventralne respiratorne grupe (VRC) u produženoj moždini korištena su tri kriterija. Prvi kriterij bile su već utvrđene stereotaksične koordinate. Drugi korišteni kriterij bio je prisutnost različitih tipova respiratornih neurona unutar VRC (inspiratornih i ekspiratornih neurona). Treći kriterij bio je karakteristični tahipnoični odgovor PNG-a na injektiranje (30-40 nl, 10 mM) glutamat agonista DLH-a (Dhomocisteična kiselina) u pBC kompleks. Hipoteza je da ako 5HT1A i 5HT2A receptori unutar pBC kompleksa i VRC pridonose promjeni ritma disanja, tada će lokalna primjena njihovih agonista postići isti učinak kao i nakon sistemske primjene. 47 Pikoinjektiranje 1, 10 i 100 μM 8OH-DPAT na pojedinačne inspiratorne (I) i ekspiratorne (E) neurone unutar pBC kompleksa smanjilo je frekvenciju izbijanja istih (Fn) za ~40%. DOI nije imao nikakav učinak na frekvenciju istih pojedinačnih respiratornih neurona unutar pBC kompleksa. Mikroinjektiranje 8OH-DPAT (200 μM), DOI (200 μM) i 5-HT (5 mM) na grupu respiratornih neurona unutar VRC od obeksa do 9 mm rostralno nije utjecalo na vrijeme inspirija (TI), vrijeme ekspirija (TE) i vršnu aktivnost freničnog živca (PPA). Sistemski (IV) administriran 8OHDPAT (19±2μg/kg) doveo je do smanjenja TI i TE za ~40%, ali nije imao učinak na PPA. Sistemski primijenjen DOI (235±48μg/kg) uzrokovao je porast TI za 17.4±5.1%, ali bez učinka na TE i PPA. Rezultati ove studije upućuju na to kako agonisti 5HT receptora sistemski administrirani mijenjaju ritam disanja. Spomenuti učinak ne postižu djelovanjem na respiratorne neurone unutar VRC(uključujući i pBC kompleks) već negdje izvan tih mjesta. Parabrahijalna i Kölliker-Fuse regija unutar mosta moguća su mjesta djelovanja agonista.Selective serotonin agonists can prevent opioid-induced depression of breathing without diminishing analgesia. When given systemically (IV), it is unclear where this effect is produced within the respiratory control network. The preBötzinger (pBC) complex has been suggested as the primary site where that happens. However, this hypothesis has not been tested pharmacologically in vivo. This study sought to determine if 5- HT1A and 5-HT2A receptors within the preBötzinger complex (pBC) and ventral respiratory column (VRC) contributed to the breathing rate response produced by IV administration of (±)-8-Hydroxy-2-dipropylaminotetralin hydrobromide (8OH-DPAT) and (±)-1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI) in vagotomized, decerebrate, paralyzed and mechanically ventilated dogs. Inhalational anesthesia was induced by mask and maintained with isoflurane at 1.5-2.5% (1-2 MAC) end-tidal concentration. The animals were monitored for signs of inadequate anesthesia such as salivation, lacrimation, and increases in blood pressure and heart rate. The animal was then decerebrated by midcollicular transection and isoflurane was discontinued. After decerebration the animal was ventilated with an air-O2 mixture and maintained in hyperoxic normocapnia (FiO2 >0.6) to exclude possible effect of anesthesia. Multibarrel micropipettes were used to record extracellular neuronal activity and to pressure eject drugs onto single neurons or into localized groups of respiratory neurons. The phrenic neurogram (PNG), cyclic TI and TE, airway CO2 concentration and systemic blood pressure were continuously displayed/recorded. We used three criteria to locate the pBC complex: 1) predetermined stereotaxic coordinates, 2) presence of a mixture of respiratory neuron subtypes within the VRC, and 3) tachypneic response of the phrenic nerve (PNG) to DLH microinjections (30-40 nl; 10 mM). Three protocolos were made. In protocol one we measured responses of pBC complex neurons to picoejection of 8OH-DPAT and DOI. Picoejection of 1, 10, and 100 μM 8OH-DPAT on single inspiratory (I) and expiratory (E) pBC neurons produced dosedependent decreases in discharge frequency (Fn) with a ceiling of ~40%. DOI had no effect on single pBC neurons. In second protocol we were looking into effects of microinjections of 8OH-DPAT, DOI and 5-HT into the VRC including the pBC on phase-timing and peak phrenic nerve activity. Microinjections of 8OH-DPAT (200 μM), DOI (200 μM) and 5-HT (5 mM) within the VRC from the obex to 9 mm rostral had no effect on I-duration (TI), E-duration (TE) and peak phrenic activity (PPA). Our last (third) protocol showed effects of IV administration of 8OH-DPAT and DOI on phase-timing and peak phrenic nerve activity.IV 8OHDPAT (19±2μg/kg) reduced TI and TE by ~40%, but had no effect on PPA. IV DOI (235±48μg/kg) increased TI by 17.4±5.1%, but had no effect on TE or PPA. The findings of this study indicate respiratory neurons in the pBC complex are inhibited maximally 40% by the 5-HT1A receptor agonist 8OH-DPAT and pBC I-neurons are depressed at lower agonist concentrations (1 μM) than E-neurons. Our findings also suggest that 5-HT2A receptors are not present or not functional on canine pBC neurons, since their discharge was unaltered by picoejection of the selective agonist DOI at relatively high concentrations (100 μM). Furthermore, our results suggest that changes in phasetiming produced by systemic administration (IV) of 8OH-DPAT and DOI are not secondary to activation of 5-HT receptors in the pBC complex. Other possible locations have been suggested. Parabrachial and Kölliker-Fuse regions of the pons are one of those