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Opioids prolong and anoxia shortens delay between onset of preinspiratory (pFRG) and inspiratory (preBötC) network bursting in newborn rat brainstems

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Abstract

Differential responses to opioids established the hypothesis that pre/postinspiratory (Pre-I) neurons of the parafacial respiratory group (pFRG) and inspiratory (Insp) neurons of the pre-Bötzinger complex (preBötC) constitute a dual brainstem respiratory center. For further analysis of pFRG/preBötC interactions, we studied in newborn rat brainstem-spinal cord preparations opioid and anoxia effects on histologically identified pFRG-driven “type-I” Insp preBötC neurons and Pre-I neurons from three distinct respiratory brainstem regions. The µ-opioid [d-Ala2,N-Me-Phe4,Gly5-ol]-enkephalin (DAMGO) slowed inspiratory-related cervical nerve bursts quantally, whereas anoxia induced nonquantal slowing and repetitive cervical bursts. DAMGO had no effect on membrane potential or input resistance of Pre-I neurons, while anoxia hyperpolarized them (~5 mV) and decreased their resistance (~30%). DAMGO prolonged the preinspiratory phase of Pre-I neuron bursting, whereas anoxia caused a shift to postinspiratory (48%) or inspiratory (22%) activity and silenced further 30% of cells. Pre-I neuron responses were not correlated with their rostrocaudal location or morphology. Neither DAMGO nor anoxia changed membrane potential of type-I neurons, but decreased their input resistance by 33% and 21%, respectively. The opposite DAMGO- and anoxia-evoked phase shifts of Pre-I neuron activity were reflected by corresponding shifts of pre/postinspiratory drive potentials in type-I neurons and, partly, by voltage-sensitive dye-imaged medullary neuronal population activities. The findings suggest that opioids presynaptically delay activation of type-I neurons as the target of drive from the pFRG to the preBötC. Contrary, anoxia seems to partly synchronize the pFRG and preBötC rhythm generators. This may enhance inspiratory and postinspiratory medullary activities for triggering multiple inspiratory motor bursts.

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Acknowledgments

This study was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture in Japan, the Canadian Institutes of Health Research (CIHR), and the Alberta Heritage Foundation for Medical Research (AHFMR). K.B. is an AHFMR Scientist. A.R. has been awarded a CIHR studentship (MFN training grant) and a AHFMR-Hotchkiss studentship.

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Authors and Affiliations

  1. Department of Physiology, Showa University School of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo, 142, Japan

    H. Onimaru

  2. Department of Physiology, 220 HMRC, University of Alberta, Edmonton, AB, Canada, T6G 2S2

    K. Ballanyi & A. Ruangkittisakul

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  1. K. Ballanyi
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  2. A. Ruangkittisakul
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Correspondence to K. Ballanyi.

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K. Ballanyi and A. Ruangkittisakul contributed equally to this work.

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Fig. S1

Morphology and location within the preBötC of type-I inspiratory neurons. The schematics in purple show the ventral aspect of the newborn rat brainstem according to the atlas in [48] with numbers indicating the distance from VIIc in mm. Open symbols represent type-I neurons that were tested for DAMGO, whereas the filled symbols show cells tested for chemical anoxia. The label “F.xx” indicates that membrane potential responses of this cell are shown in the corresponding figure in the main paper. Asterisks indicate axons. (DOC 342 kb)

Fig. S2

Morphology and location of Pre-I neurons. The schematics in purple show the ventral aspect of the newborn rat brainstem according to the atlas in [48] with numbers indicating the distance from VIIc in mm. Open symbols indicate Pre-I neurons that were tested for DAMGO, whereas the filled symbols represent cells tested for chemical anoxia. Different filled symbols are used to indicate distinct response types during the early (<10 min) and later (20–30 min) phase of anoxia. The label “F.xx.” indicates that membrane potential responses of this cell are shown in the corresponding figure in the main paper. Asterisks indicate axons. (DOC 399 kb)

Fig. S3

Opposite respiratory phase shifts associated with the depression of breathing in newborn mammals by opioids and anoxia. As shown in the box in the center, inspiratory-related motor output is recorded, e.g., from the C4 root in the isolated newborn rat brainstem-spinal cord model. Such C4 nerve bursting is generated within inspiratory preBötC networks that are presumably driven by rhythmogenic Pre-I neurons constituting the pFRG. Specifically, pFRG neurons appear to provide preinspiratory drive to type-I Insp neurons that may play an important role in triggering the onset of preBötC bursting. The preBötC and pFRG networks are mutually synaptically coupled as evident from inspiratory-related inhibition of Pre-I neurons via receptor-coupled anion channels. The upper box shows that opioids notably prolong the time period of preinspiratory bursting in Pre-I neurons and concomitantly shorten their postinspiratory bursting. Assuming that the activity of the pFRG starts the respiratory cycle, this effect may be due to delayed generation of inspiratory motor output. This delay may result from impaired synaptic transmission between pFRG and type-I Insp neurons as suggested by a greatly reduced slope of subthreshold drive potentials in type-I Insp neurons. Opioids also decrease the amplitude and duration of the C4 burst, but increase the time to its peak. According to this hypothetical scenario, a C4 motor burst is not elicited when the depressed transmission involving both pre- and postsynaptic μ-opiate receptors does not reach the threshold for activation of a preBötC burst. As shown in the lower box, anoxia decreases the delay between the onset of pFRG and preBötC bursting and thus almost synchronizes the presumably dual respiratory rhythm generator. Accordingly, the preinspiratory drive potential is shortened in type-I Insp neurons, whereas Pre-I neurons show three different responses to sustained (10–20 min) anoxia. In 48% of cells, the bursting pattern shifts toward the postinspiratory phase with concomitant depression of the duration of preinspiratory activity and the strength of postinspiratory bursting. In 22% of Pre-I neurons, the activity pattern changes to inspiratory bursting, whereas the remaining 30% are “functionally inactivated” because of greatly depressed drive potentials. Possibly because of the transformation of some Pre-I neurons into Insp cells and the concomitant depression of synaptic inhibition, C4 burst amplitude increases by ~50%, while the time to peak decreases, resulting in sharper burst resembling anoxic gasps in vivo. Not shown in this schema are hypothetic medullary neurons, which develop pronounced postinspiratory activity during anoxia as indicated by findings from voltage-sensitive dye-imaging. The overall enhanced inspiratory/postinspiratory medullary activity during anoxia may be responsible for triggering inspiratory motor bursts doublets or triplets. For more information, see text. (DOC 266 kb)

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Ballanyi, K., Ruangkittisakul, A. & Onimaru, H. Opioids prolong and anoxia shortens delay between onset of preinspiratory (pFRG) and inspiratory (preBötC) network bursting in newborn rat brainstems. Pflugers Arch - Eur J Physiol 458, 571–587 (2009). https://doi.org/10.1007/s00424-009-0645-3

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