Control of Breathing & Airway Defense Seminar Series - Shared screen with speaker view
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To everyone, fire away with questions. Postdoctoral fellow Ada Varga and graduate student Sandy Saunders from the Levitt laboratory are on line to answer short questions.
Christopher Del Negro
In a nutshell, what is inductance plethysmography? How is it like/unlike whole-body barometric plethysmography?
Inductance plethysmography uses a pulse transducer (strap) that is fixed against the abdomen to measure rate and tidal volume.
Q: are these recordings in TTX to help address the ongoing debate in many respiratory area about pre- vs postsynaptic site of action
No, the cells were not isolated in TTX.
Q: desensitization can occur via a number of processes from changes in the gating properties through to receptor internalization. In LC neurons does internalization contribute?
Q: If you did not use TTX, what is the basis for the conclusion that the effects on KF neurons are postsynaptic
Erica will address the two questions above in the discussion period.
Q: Any change in basal breathing in MOR-cKO mice?
We did not find a significant change in their baseline breathing.
What was the duration of the apneas that she just showed with morphine?
How about tidal volume? The effect was only in respiratory frequency?
Q:What was the duration of the apneas that she just showed with morphine? Sorry- forgot to add the Q!
Our cut off for apneas was 1 second, but most of them lasted multiple seconds.
We didn't measure tidal volume, because in our hands the head-out pleth technique was not appropriate. The seal dividing the head and body chambers was a flexible membrane that changed during the course of the experiment making tidal volume measurements unreliable.
Have you tried targeting known mechanisms of mu receptor desensitization (e.g., PKC, arrestin) in the KF as a means of preventing opioid induced respiratory depression?
Erica will address this question during the discussion at the end.
Q: The genetic approach to distinguish pre vs postsynaptic mechanisms confuses me in that the transfection of the KF neurons will remove postsynaptic receptors in KF neurons but this will also remove presynaptic receptors from KF axon terminals (e.g., if KF neurons that project to preBotC express opioid receptors on their axon terminals, these opioid receptors are presynaptic to preBotC neurons. So now any change in the effect of exogenous opioids on ventilation could be due to lack of postsynaptic KF opioid receptors or presynaptic opioid receptors onto preBotC neurons). What am I missing?
Q: did fentanyl reduce perfusion pressure in the WHBP, as reduced perfusion can also result in an apneustic breathing pattern that looks very similar
Christopher Del Negro
Q: Greg Funk’s point is well taken, µOR knock-out in the KF would remove µOR expressing terminals in the preBötC too.
(Q) How can you interpret your results from in situ experiments suggesting that opioid-induced apnea is due to opioid effect on KF if complete inhibition of KF in in situ preparation perturbs, but does not stop breathing rhythm? (Bautista & Dutschmann, 2014, Neuroscience)
Fentanyl often (but not always) reduced perfusion pressure. To attempt to counter this, we have tried increasing perfusion pressure (via pump) during fentanyl exposure and apnea persists.
Yes, both of you are right. All MORs were deleted from KF neurons, somatic as well as the ones on axon terminals.
With high morphine doses, what do you think is responsible for the part of the respiratory rate reduction that remains after KF MOR deletion?
Agonist or antagonist injection into the KF would affect the somatic MORs and MORs on incoming axon terminals (for instance the ones on preBotC projection neurons). But with the MOR removal technique we were able to remove the receptors from KF neurons only. I hope this helps.
Opioid-induced apnea includes inhibition of the entire ponto-medullary respiratory circuit. Antagonist into the KF can merely counter this rate depression, through presumable excitatory drive to medullary centers. Erica has injected opioids into the KF during her post doctoral work. The effect of opioids into the KF was loss of the post-inspiratory output and increase in inspiratory duration of phrenic and vagal output.
What are the phenotypes of these KF neurons?
Erica’s lab is filled with talented wonderful people and it is clear that she has inspired them to do amazing and productive work in a critically important area - congratulations Erica
phenotype in this context refers to rhythmic firing pattern i.e. inspiratory, expiratory, post-inspiratory, phase-spanning etc.
but I think it is a future study. Tks
[q] What would you predict would happen if you did the WHBP apnea experiments with the PreBotc instead of the KF as your injection site?
Q:with 1s cut for apnea, you are not sub-estimation longer expiration?(for apneas calculation usually, it is used the time of 2 respiratory cycles, it is around 2s - considering breathing frequency of 90cps)
Christopher Del Negro
yeap I got it
Q Can you explain the effect or interaction of sleep state on any of these mechanisms of OIRD?
Great presentation. How do you distinguish the KF neurons from those in the lateral crescent of the PB with similar connections?
Q) Can you separete spontaneous or post sight apneas? since the mechanisms that regulate they are diferente..
Morphine wipes out REM sleep in mice. At least as far as we can see after 10 mg/kg
Well done to both Ericas! Thank you all for coming.
Not trying to rush things!
Post-inspiratory activity is very sensitive to mu-opioid receptor agonists, with major implications for upper airway control. Could you comment on mechanisms for the high sensitivity of post-inspiratory activity.
Thank you Erica! Nice talk
Excellent talk Erica!
If perfusion pressure changes this changes brain PCO2 and central chemoreceptor drive.
Excellent talk Erica! Thank you!
Q: Do we know if metabolic capacity can confound these outcomes? In other words, if mitochondrial function or antioxidant capacity are improved, does it mitigate or augment these respiratory measures?
Thanks Dr. Levitt for such a nice talk!
Thanks a lot!