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Control of Breathing & Airway Defense Seminar Series - Shared screen with gallery view
Kate Casey-Sawicki
27:09
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Kate Casey-Sawicki
27:26
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Kate Casey-Sawicki
27:46
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Christopher Del Negro
43:50
Q: is the virally delivered Cre expressing in all preBötC neurons or is it specific for Vglut2+ neurons in preBötC?
Iris Bachmutsky
45:23
The cre is expressed in all preBotC neurons, nonspecifically
Iris Bachmutsky
45:41
(really all neurons in the prebotc region)
Caroline Szujewski
45:59
What is the dose of morphine used in these experiments?
Nathan Baertsch
46:10
Q: How specific were the viral injection for KF vs. the parabrachial region as a whole?
Seva Polotsky
46:15
How did you express cre in all pre-Botz neurons?
Iris Bachmutsky
46:35
20mg/kg
Fernando Peña
48:01
Was Oprm1 expressed in glial cells?
Iris Bachmutsky
48:15
For the KF-- The virus infected cells throughout the parabrachial region, but since the cre is deleting the MOR, and the MOR is expressed fairly specifically in the KF, the functional effect is presumably relatively limited
Richard Wilson
48:25
Was the NA spared?
Iris Bachmutsky
49:04
how did you express cre: virus expressed cre fused to GFP, under a generic promotor
Iris Bachmutsky
49:34
NA spared- no, it was not. We're conducting follow up experiments to separate its effect through intersectional genetics.
Richard Wilson
50:35
thanks…great work btw!
Iris Bachmutsky
50:47
thank you!
jack feldman
51:49
Cell counts for effects of 50-140 neurons (as well as CDN experiments) are from slice experiments. Any data from intact mice?
Iris Bachmutsky
53:55
jack: if you mean whether we have shown that vglut2 neurons play a preferred role in sensitivity to opioids in the pbc in vivo (therefore fewer neurons likely involved), we haven't finished gathering that dataset yet, but we're working on it.
Christopher Del Negro
55:28
I think the issue is whether there is evidence in vivo for destroying 50-140 being able to stop breathing
Paul Wei
56:27
follow up to iris: in addition to vglut2 neurons, we’re also working on deleting oprm1 from other, much smaller molecular subsets of prebotc or nearby neuron, and see whether we can see this effect in vivo.
Iris Bachmutsky
56:55
CDN: Right, that is based on an assumption that the in vitro effect replicates in vivo, which we have yet to fully prove. So, tbd but its an intriguing idea.
Nathan Baertsch
01:03:01
Q: How quantifiable, in terms of absolute cell counts, is the RNAseq data? Would interpreting this data in terms of the relative proportions of cells be more appropriate?
Paul Wei
01:04:23
Yes, Kevin wasn’t super clear, but the RNAseq result was used to look at proportion of cells that express oprm1, not absolute numbers .
Erica Levitt
01:05:17
Q: You also showed that inhibitory neurons in the preBotC area expressed MOR transcript in your RNAseq data. What do you think those MOR-expressing inhibitory neurons are doing?
Paul Wei
01:09:13
Super interesting question and the short answer is we don’t really know! We’re hoping to isolate this in vivo and see if there may be differences in vivo.
Luciane Gargaglioni
01:09:44
Are these cry signals are USV waves?
Iris Bachmutsky
01:09:51
EL about inhib neurons: I'd love to know! But to know that I'd have to know more about how and when endogenous opiates act on respiration. It's interesting the net effect is still clearly to depress breathing, even though its 50/50 inhib, excitabory.
Paul Wei
01:10:13
LG: Yes they are USVs
Luciane Gargaglioni
01:10:18
Tks
Thiago Moreira
01:13:39
In terms of evolution, why do respiratory areas in the brainstem express opioids receptors?
Iris Bachmutsky
01:15:44
TM in regards to evolution: Unfortunately, I don't know. There are some theories about the rush of opiates at birth and the transition to breathing air etc, but I dont think theres a clear answer out there yet.
Jan Ramirez
01:16:50
This is exactly the location of PiCo the postinspiratory complex
Jan Ramirez
01:17:07
And PiCo is excitatory
Richard Kinkead
01:19:19
That is a very good question! Vasilakos et al (2005) showed that in frogs, morphine depresses fictive air breathing but not motor output related to gill breathing.
Paul Wei
01:19:46
There are definitely anatomical overlap between this area and PiCo. However, there are some molecular and electrophysiological differences between these neurons and PiCo. Perhaps Kevin can discuss some of these differences in the Q&A.
Jan Ramirez
01:19:46
These lesions would have also lesioned PiCo
Jan Ramirez
01:20:02
Yes, this should be discussed
Jan Ramirez
01:20:25
The preBötC contains also different molecularly defined neurons
Luciane Gargaglioni
01:20:41
How about the periaqueductal gray that is considered the gating center for vocal behavior?
Jan Ramirez
01:21:08
PiCo could also oscillate at a higher frequency than PreBotC
Jan Ramirez
01:22:03
… and it localized exactly with PiCo
Kimberly Iceman
01:23:14
Hulsmann showed that the start of USVs shifts from the expiratory peak to the zero flow point/inspiration peak during the ~2nd postnatal week. This suggests that generation of USVs transitions from a dependency on active expiration to the ability to generate with post-inspiratory airflow. How does this correspond with your data/model, and have you looked at this age?
Nathan Baertsch
01:23:16
The preBotC is considered the inspiratory rhythm generator, produces multiple behaviors - eupnea sighs and gasps, and is heterogeneous - containing excitatory, inhibitory, pacemaker neurons, etc. Why then should the region described here that overlaps with PiCo and is for postinspiratory vocalizations be considered a distinct region from PiCo?
Christopher Del Negro
01:23:38
Q: The former slide in transverse section showed the putative iRO just dorsal to preBötC, but the anterior-posterior location should not be at the level of preBötC but rather more rostral at the level of BötC
Jan Ramirez
01:23:48
PiCo also oscillates independent of preBotC
Swen Hülsmann
01:23:51
Q: in mice P8-12, USVs do not require active expiration. Does lesioning the rv-iRF effect the cries in older mice as well?
Paul Wei
01:25:05
Re: PAG - we’re interested in the connection from the PAG! It likely serves as gate, but don’t have definitively evidence in neonates.
Luciane Gargaglioni
01:25:40
Thanks Paul
Paul Wei
01:25:41
SH: We haven’t looked at older mice but it’s an interesting question.
Nicholas Burgraff
01:26:23
What happens to the recorded activity from this region when you add norepinephrine?
Christopher Del Negro
01:26:38
Q: the behavioral data seem to show inspiratory airflow, which Kevin called “mini” inspirations. But how can postI laryngeal adduction give rise to “mini” inspiration. I don’t understand how it would reverse the airflow from E to I.
Jan Ramirez
01:26:42
Beautiful talk Kevin, but why call it iRO or rv-IRF if this exact area (PiCo) has previously been described to be critical for post inspiration
Paul Wei
01:29:21
CDN: I’m not sure if I’m understanding the question correctly, but the “mini-inspirations” are each driven by a inspiratory motor program, which are each followed by post laryngeal adduction that produces the sound.
Swen Hülsmann
01:29:50
PW: The inhibition from the preBöTC should be GABAergic, since you see the USV-calls also in GlyT2-KO mice
Nathan Baertsch
01:31:23
are preBotC pacemakers a different region than the preBotC?
Christopher Del Negro
01:31:41
PW: So, I thought that iRO was engaging the inspiratory pattern. If iRO is engaging the post-I motor program then what part of this putative circuit engages the INSP program
Jan Ramirez
01:32:11
PiCo contains NOT ONLY. Cholinergic neurons
Nicholas Burgraff
01:34:07
Wouldn’t pacemaker function just show that pico contains intrinsically bursting neurons?
Caroline Szujewski
01:34:09
I may have missed this, but if not-Are the iRO neurons sensitive to opioids?
Kaiwen Kam
01:35:00
Q: In your lesion experiments, did you test for any changes in swallowing or other post-inspiratory motor behaviors?
Jan Ramirez
01:36:25
The region that controls active expiration is primarily defined by the location in the RTN - why do you disregard PiCo if it is exactly the same as “your” region?
Sandy Saunders
01:36:57
Just a comment. From your data traces, it seems a high amplitude (higher than breaths without calls and peak around late I) inspiratory breath occurs prior to the calls. Late-Inspiratory pontine (dorsolateral pons) neuronal activity could be important for initiation of these vocalizations (laryngeal recruitment).
Nathan Baertsch
01:37:00
none of the regions are defined by a single phenotype of neuron
Christopher Del Negro
01:37:27
I think we should be careful about what is or is not PiCo. There is only 1 paper characterizing the putative PiCo region and at least 2 studies that looked for PiCo but didn’t find it
Jan Ramirez
01:37:50
Sorry Chris there are several PiCO paper by now
Nathan Baertsch
01:39:34
I think Kevin's great work here is showing amazing data, and in fact supports the existence of PiCo quite nicely. The only quibble seems to be regarding nomenclature...
Nathan Baertsch
01:40:20
Nice talk Kevin!
Kaiwen Kam
01:40:21
Great job, Kevin!!!
Swen Hülsmann
01:40:31
thanks
Christopher Del Negro
01:40:32
AWESOME talk Kevin
Aaron Betts
01:40:41
Thank you for the great talk!
Nicholas Burgraff
01:40:53
Thanks Kevin!