Activating convergent signal pathways in respiratory neurons of the ventral medullary group

Abstract

Opiates are known to disrupt the respiratory rhythm by binding to μ- and δ-type opioid receptors of respiratory neurons within the ventral medullary group and by activating signal pathways that induce depression of neuronal excitability and synaptic interaction. In previous experiments we demonstrated that opioid depression of respiration can be treated by a variety of drugs that increase intracellular cAMP levels. In the present study, we investigated how activation of μ-type opioid receptors can be counteracted and respiratory depression be treated by activation of convergent signal pathways targeting the same second messenger systems of the neurons. Our starting point was the observation that a compensatory effect can be achieved with Buspirone, a drug purported to activate 5-HT1A receptors and, consequently, to reduce intracellular cAMP levels [1]. If these effects were confirmed, this observation would indicate a profound non-specificity of the 5-HT-1A directed drug. The task then would be to identify the serotonin receptor isoforms responsible. In various preparations, including the anaesthetized in vivo cat, the perfused mouse or rat brain stem and the brain stem slice of the mouse or rat, we performed current and voltage clamp measurements with fine tipped or patch electrodes to measure electrophysiological parameters. RT-PCR methods were applied to identify the mRNA encoding for serotonin receptor isoforms, while immunocytochemical techniques were used to verify receptor expression. We verified the stabilizing effect of cAMP [2,3] and confirmed the protective effect of 5-HT1A-receptor agonists 8-OHDPAT and Buspirone against opioid depression of neural respiratory activity [4]. A detailed inspection of the results obtained with the commonly used 5-HT1A-agonists, 8-OH-DPAT and Buspirone, indicated ambiguous effects of these drugs. Our conclusion was that these drugs probably act on other serotonin receptor isoforms which are expressed in addition to the known 5-HT1A and 5-HT2A subtypes. Therefore, we developed two novel antibodies and demonstrated the expression of 5-HT4 and 5-HT7 receptors in neurons of the VRG and the hypoglossal nuclei. These immunocytochemical findings were verified by quantitative RT-PCR analysis of the VRG region and confirmed in single cell RT-PCR analysis on identified respiratory neurons. Finally, we demonstrated that these findings provide a basis for novel strategies for the treatment of respiratory depression induced by opioids. The same strategy seems to be efficient in the treatment of respiratory disturbances induced by barbiturates.