Neuron populations use variable combinations of short-term feedback mechanisms to stabilize firing rate.

Neurons tightly regulate firing rate and a failure to do so leads to multiple neurological disorders. Therefore, a fundamental question in neuroscience is how neurons produce reliable activity patterns for decades to generate behavior. Neurons have built-in feedback mechanisms that allow them to monitor their output and rapidly stabilize firing rate. Most work emphasizes the role of a dominant feedback system within a neuronal population for the control of moment-to-moment firing. In contrast, we find that respiratory motoneurons use 2 activity-dependent controllers in unique combinations across cells, dynamic activation of an Na+ pump subtype, and rapid potentiation of Kv7 channels. Both systems constrain firing rate by reducing excitability for up to a minute after a burst of action potentials but are recruited by different cellular signals associated with activity, increased intracellular Na+ (the Na+ pump), and membrane depolarization (Kv7 channels). Individual neurons do not simply contain equal amounts of each system. Rather, neurons under strong control of the Na+ pump are weakly regulated by Kv7 enhancement and vice versa along a continuum. Thus, each motoneuron maintains its characteristic firing rate through a unique combination of the Na+ pump and Kv7 channels, which are dynamically regulated by distinct feedback signals. These results reveal a new organizing strategy for stable circuit output involving multiple fast activity sensors scaled inversely across a neuronal population.

Activation of respiratory-related bursting in an isolated medullary section from adult bullfrogs.

Breathing is generated by a rhythmic neural circuit in the brainstem, which contains conserved elements across vertebrate groups. In adult frogs, the 'lung area' located in the reticularis parvocellularis is thought to represent the core rhythm generator for breathing. Although this region is necessary for breathing-related motor output, whether it functions as an endogenous oscillator when isolated from other brainstem centers is not clear. Therefore, we generated thick brainstem sections that encompass the lung area to determine whether it can generate breathing-related motor output in a highly reduced preparation. Brainstem sections did not produce activity. However, subsaturating block of glycine receptors reliably led to the emergence of rhythmic motor output that was further enhanced by blockade of GABAA receptors. Output occurred in singlets and multi-burst episodes resembling the intact network. However, burst frequency was slower and individual bursts had longer durations than those produced by the intact preparation. In addition, burst frequency was reduced by noradrenaline and µ-opioids, and increased by serotonin, as observed in the intact network and in vivo. These results suggest that the lung area can be activated to produce rhythmic respiratory-related motor output in a reduced brainstem section and provide new insights into respiratory rhythm generation in adult amphibians. First, clustering breaths into episodes can occur within the rhythm-generating network without long-range input from structures such as the pons. Second, local inhibition near, or within, the rhythmogenic center may need to be overridden to express the respiratory rhythm.

Fentanyl effects on respiratory neuron activity in the dorsolateral pons.

Opioids suppress breathing through actions in the brainstem, including respiratory-related areas of the dorsolateral pons, which contain multiple phenotypes of respiratory patterned neurons. The discharge identity of dorsolateral pontine neurons that are impacted by opioids is unknown. To address this, single neuronal units were recorded in the dorsolateral pons of arterially perfused in situ rat preparations that were perfused with an apneic concentration of the opioid agonist fentanyl, followed by the opioid antagonist naloxone (NLX). Dorsolateral pontine neurons were categorized based on respiratory-associated discharge patterns, which were differentially affected by fentanyl. Inspiratory neurons and a subset of inspiratory/expiratory phase-spanning neurons were either silenced or had reduced firing frequency during fentanyl-induced apnea, which was reversed upon administration of naloxone. In contrast, the majority of expiratory neurons continued to fire tonically during fentanyl-induced apnea, albeit with reduced firing frequency. In addition, pontine late-inspiratory and postinspiratory neuronal activity were absent from apneustic-like breaths during the transition to fentanyl-induced apnea and the naloxone-mediated transition to recovery. Thus, opioid-induced deficits in respiratory patterning may occur due to reduced activity of pontine inspiratory neurons, whereas apnea occurs with loss of all phasic pontine activity and sustained tonic expiratory neuron activity.NEW & NOTEWORTHY Opioids can suppress breathing via actions throughout the brainstem, including the dorsolateral pons. The respiratory phenotype of dorsolateral pontine neurons inhibited by opioids is unknown. Here, we describe the effect of the highly potent opioid fentanyl on the firing activity of these dorsolateral pontine neurons. Inspiratory neurons were largely silenced by fentanyl, whereas expiratory neurons were not. We provide a framework whereby this differential sensitivity to fentanyl can contribute to respiratory pattern deficits and apnea.

Compensatory changes in GABAergic inhibition are differentially expressed in the respiratory network to promote function following hibernation.

The respiratory network must produce consistent output throughout an animal's life. Although respiratory motor plasticity is well appreciated, how plasticity mechanisms are organized to give rise to robustness following perturbations that disrupt breathing is less clear. During underwater hibernation, respiratory neurons of bullfrogs remain inactive for months, providing a large disturbance that must be overcome to restart breathing. As a result, motoneurons upregulate excitatory synapses to promote the drive to breathe. Reduced inhibition often occurs in parallel with increased excitation, yet the loss of inhibition can destabilize respiratory motor output. Thus, we hypothesized that GABAergic inhibition would decrease following hibernation, but this decrease would be expressed differentially throughout the network. We confirmed that respiratory frequency was under control of GABAAR signaling, but after hibernation, it became less reliant on inhibition. The loss of inhibition was confined to the respiratory rhythm-generating centers: non-respiratory motor activity and large seizure-like bursts were similarly triggered by GABAA receptor blockade in controls and hibernators. Supporting reduced presynaptic GABA release, firing rate of respiratory motoneurons was constrained by a phasic GABAAR tone, but after hibernation, this tone was decreased despite the same postsynaptic receptor strength as controls. Thus, selectively reducing inhibition in respiratory premotor networks promotes stability of breathing, while wholesale loss of GABAARs causes non-specific hyperexcitability throughout the brainstem. These results suggest that different parts of the respiratory network select distinct strategies involving either excitation (motoneurons) or inhibition (rhythm generator) to minimize pathological network states when engaging plasticity that protects the drive to breathe.

Understanding and countering opioid-induced respiratory depression.

Respiratory depression is the proximal cause of death in opioid overdose, yet the mechanisms underlying this potentially fatal outcome are not well understood. The goal of this review is to provide a comprehensive understanding of the pharmacological mechanisms of opioid-induced respiratory depression, which could lead to improved therapeutic options to counter opioid overdose, as well as other detrimental effects of opioids on breathing. The development of tolerance in the respiratory system is also discussed, as are differences in the degree of respiratory depression caused by various opioid agonists. Finally, potential future therapeutic agents aimed at reversing or avoiding opioid-induced respiratory depression through non-opioid receptor targets are in development and could provide certain advantages over naloxone. By providing an overview of mechanisms and effects of opioids in the respiratory network, this review will benefit future research on countering opioid-induced respiratory depression. LINKED ARTICLES: This article is part of a themed issue on Advances in Opioid Pharmacology at the Time of the Opioid Epidemic. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v180.7/issuetoc.

Kölliker-Fuse/Parabrachial complex mu opioid receptors contribute to fentanyl-induced apnea and respiratory rate depression.

Overdoses caused by the opioid agonist fentanyl have increased exponentially in recent years. Identifying mechanisms to counter progression to fatal respiratory apnea during opioid overdose is desirable, but difficult to study in vivo. The pontine Kölliker-Fuse/Parabrachial complex (KF/PB) provides respiratory drive and contains opioid-sensitive neurons. The contribution of the KF/PB complex to fentanyl-induced apnea was investigated using the in situ arterially perfused preparation of rat. Systemic application of fentanyl resulted in concentration-dependent respiratory disturbances. At low concentrations, respiratory rate slowed and subsequently transitioned to an apneustic-like, 2-phase pattern. Higher concentrations caused prolonged apnea, interrupted by occasional apneustic-like bursts. Application of CTAP, a selective mu opioid receptor antagonist, directly into the KF/PB complex reversed and prevented fentanyl-induced apnea by increasing the frequency of apneustic-like bursting. These results demonstrate that countering opioid effects in the KF/PB complex is sufficient to restore phasic respiratory output at a rate similar to pre-fentanyl conditions, which could be beneficial in opioid overdose.