Morphine and synthetic 5-opioid receptor (5OR) analogs such as fentanyl and remifentanil (remi) are highly effective analgesics used to treat severe acute and chronic pain. Profound respiratory depression (bradypnea, apnea) can occur at clinically relevant plasma concentrations. Whether these effects are due to depression of highly opioid sensitive respiratory regions or dispersed over many synapses remains unresolved. Finding highly opioid sensitive targets is an important step in designing strategies to prevent respiratory depression during opioid analgesia. Our studies indicate that opioid-induced bradypnea does not result from activation of 5ORs in the preBvtzinger Complex (pBC), the putative locus for rhythm generation. Preliminary studies suggest that 5ORs on/near pontine respiratory group (PRG) neurons in parabrachial/ Kvlliker-Fuse nuclei (PB- KF region) are targets. Our working hypothesis is that clinical concentrations of systemic 5-opioids act at 5ORs in the PB-KF region to produce bradypnea by either direct activation of 5ORs on subtypes of PRG neurons and/or indirectly via excitatory and/or inhibitory synaptic inputs by other opioid-sensitive pontine neurons. These subtypes of PRG neurons and pulmonary stretch receptors (PSRs) inputs control the medullary pBC/Bvtzinger complex (BC) neurons responsible for respiratory phase timing/switching. Also, activation of specific amine receptors in the PB-KF region may reverse opioid-induced respiratory depression. To address these hypotheses, the following specific objectives will be pursued: Objective 1: To locate the region in the PB-KF area that produces opioid-induced bradypnea via DAMGO (5OR agonist) microinjections by monitoring changes in respiratory phase durations from the phrenic neurogram (PNG). Naloxone (NAL;opioid antagonist) microinjections during IV remi will be used to determine if the same 5ORs produce bradypnea. Histochemical methods will be used to identify regions with high densities of 5OR immunoreactivity (IR) to confirm the functionally localized regions. Objective 2: To determine which types of PRG neurons are most susceptible to IV remi and PB-KF regional DAMGO depression. Objective 3A: To determine if the IV remi depression of PRG neuronal discharge is due to postsynaptic activation of 5ORs, indicated by NAL reversal picoejected on single neurons. 3B: For nonreversed PRG neurons, to determine if they possess 5ORs via picoejection of DAMGO. Objective 4A: To determine if changes in glutamatergic excitation mediated by NMDA and AMPA receptors and/or GABAergic and glycinergic inhibition are involved in IV remi depression of PRG neurons. Picoejection of selective antagonists on single PRG neurons before and during IV remi bradypnea will be used. Objective 5. To determine if 5HT1A, D1 dopamine and 12 adrenergic receptors are present on subtypes of PRG neurons, to serve as therapeutic targets to counteract 5OR-induced depression. Systemic IV infusions of remi will be used to produce bradypnea in an in vivo decerebrate canine model. Phrenic nerve activity will be used to measure I- and E-phase duration. Multibarrel micropipettes will be used to record the discharge activity of single PRG neurons while ejecting neuroactive agents. A 16-electrode array probe (NeuroNexus) will be used to obtain simultaneous recordings of multiple PRG neurons before and during remi-induced bradypnea. Responses to antidromic activation and PSR inputs will be used to classify PRG neurons. These studies will answer whether 5ORs in the PB-KF region mediate the depression of breathing frequency produced by systemic 5-opioids at clinical concentrations, will identify subtypes of opioid sensitive PRG neurons and answer if effects are direct and/or indirect. In addition, these studies will determine whether receptors for aminergic neuromodulators on PRG neurons offer a therapeutic target to minimize opioid-induced depressant effects. These studies will also provide important new information on the functional roles of PRG neurons and the contribution of specific neurotransmitters/modulators in the generation of discharge patterns of various PRG neurons in vivo and new insights into phase-timing mechanisms.

Public Health Relevance

The vital importance of adequate respiratory function in the face of the polymorbidity of our veterans cannot be overemphasized. Many pathologic conditions that are common to the veteran population compromise respiratory function and include: diseases of the airways, lungs, and cardiovascular system, injuries to the head, brain, and cervical spinal cord, brain tumors, central and obstructive sleep apneas associated with the metabolic syndrome and hypertension. In addition, this patient population has a frequent need for potent opioid analgesics, be it for perioperative pain control or chronic pain from trauma, degenerative diseases or cancer. The proposed studies will contibute to the detailed knowledge and understanding of the control of breathing and of the neurophysiology and neuropharmacology of various respiratory neuron groups. This type of information is a necessary prerequisite for designing specific medications and therapeutic strategies that allow adequate pain control by opioids while minimizing respiratory depression.

National Institute of Health (NIH)
Veterans Affairs (VA)
Non-HHS Research Projects (I01)
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Neurobiology A (NURA)
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Clement J. Zablocki VA Medical Center
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Zuperku, Edward J; Stucke, Astrid G; Krolikowski, John G et al. (2018) Inputs to medullary respiratory neurons from a pontine subregion that controls breathing frequency. Respir Physiol Neurobiol :
Zuperku, Edward J; Stucke, Astrid G; Hopp, Francis A et al. (2017) Characteristics of breathing rate control mediated by a subregion within the pontine parabrachial complex. J Neurophysiol 117:1030-1042
Miller, Justin R; Zuperku, Edward J; Stuth, Eckehard A E et al. (2017) A Subregion of the Parabrachial Nucleus Partially Mediates Respiratory Rate Depression from Intravenous Remifentanil in Young and Adult Rabbits. Anesthesiology 127:502-514
Radocaj, Tomislav; Mustapic, Sanda; Prkic, Ivana et al. (2015) Activation of 5-HT1A receptors in the preBötzinger region has little impact on the respiratory pattern. Respir Physiol Neurobiol 212-214:9-19
Stucke, Astrid G; Miller, Justin R; Prkic, Ivana et al. (2015) Opioid-induced Respiratory Depression Is Only Partially Mediated by the preBötzinger Complex in Young and Adult Rabbits In Vivo. Anesthesiology 122:1288-98
Zuperku, Edward J; Prkic, Ivana; Stucke, Astrid G et al. (2015) Automatic classification of canine PRG neuronal discharge patterns using K-means clustering. Respir Physiol Neurobiol 207:28-39
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Prkic, Ivana; Mustapic, Sanda; Radocaj, Tomislav et al. (2012) Pontine ?-opioid receptors mediate bradypnea caused by intravenous remifentanil infusions at clinically relevant concentrations in dogs. J Neurophysiol 108:2430-41