Study of a putative novel respiratory center – Phox2RESP
Characterization of a new respiratory center
This project aims at deciphering the function of a new group of putative respiratory neurons in the hindbrain of the mouse: peri-trigeminal neurons (Peri5). Using genetic tools derived from developmental studies, we will map the connectivity of the Peri5 neurons, target specific mutations to them and characterize their function in reduced ex vivo and in vivo preparations.<br /><br />
General objective of the project and the main issues raised
This project consists of elucidating the function of a group of neurons, defined genetically in the posterior brain of mammals. More specifically, our study will target a group of neurons in the pons, probably involved in respiration. In previous work on the role of the retrotrapezoid nucleus (RTN) in respiratory rhythmogenesis and in the chemoreflex (ie the respiratory response to hypercapnia), we have identified a small population of neurons with the same signature molecule that the RTN (expressing transcription factors Phox2b and Atoh1) and a related localization (around a branchiomotor nucleus, in this case the motor nucleus of the trigeminal rather than the facial), which we call peri5 neurons. Several arguments suggest that these neurons may be involved in respiration and that, at the very least, their disappearance, combined with that of the NTN, could lead to neonatal mortality in patients affected by congenital central hypoventilation syndrome (CCCHS) by a mutation in PHOX2B) or mouse models of this disease. The project tries to elucidate the function of these neurons. Thanks to the intersectional transgenesis technique using the Flp and Cre recombinases, we will mark these neurons specifically, measure their spontaneous activity, map their conneome by viral tracing, destroy them by conditional mutations, interfere with their activity by chemogenetics in the newborn and the adult, and study the consequences of his manipulations on breathing and oro-facial rhythms. Moreover, thanks to their specific molecular signature (coexpression of Phox2b + and Atoh1 +) shared only with RTN, we will look for the putative homologues of RTN and peri5 in fish, as the first step in an evolutionary study of respiratory rhythmogenesis in l the cellular scale.
-R2Flpo line in construction at the Clinique de la souris
-Crosses to obtain Phox2b::FLPo;Atoh1FRTCre;ROSAloxSTOPlox-GCaMP6f mice
-Crosses to obtain Phox2b::FLPo;Atoh1FRTCre; Ai32 (or ;Ai35) animals
-Anterograde tracing of the peri5
-Making of an antibody against atoh1a/b. ISH with Phox2b and atoh1a/b on zebrafish brains.
-Validation of the R2Flpo line and crosses to obtain R2::FLPo;Atoh1FRTCre;Phox2blox/lox
-R2::FLPo;Atoh1FRTCre;Phox2b27Alacki and R2::FLPo;Atoh1FRTCre;VGlut2lox/lox mice.
-Defining the pattern of spontaneuous activity of peri5 cells in Phox2b::FLPo;Atoh1FRTCre;ROSAloxSTOPlox-GCaMP6f mice
-Holographic stimulation of Phox2b::FLPo;Atoh1FRTCre; Ai32 (or ;Ai35) preparations
-Set up of retrograde tracing from the peri5
-Tests of promoter fragments in transgenic zebrafish
-In vivo and in vitro analysis of R2::FLPo;Atoh1FRTCre;Phox2blox/lox
R2::FLPo;Atoh1FRTCre;Phox2b27Alacki and R2::FLPo;Atoh1FRTCre;VGlut2lox/lox mice.
-Chemogenetic silencing of Phox2b::FLPo;Atoh1FRTCre;RosaloxSTOPloxhM4Di-DREADD
-Retrograde tracing from the peri5 and identification of presynaptic cells.
-Tests of promoter fragments in transgenic zebrafish and creation of a Gcamp6 line
- The construction of the R2::FLPo transgenics has been delayed.
- We find that R2::cre;Phox2b27AlaCKI mutant pups, which have a damaged Peri5 nucleus, present at birth with a breathing impairment: ventilation is reduced by half through reduction of tidal volumes, with a normal breathing frequency.
- We have now produced Phox2bFLPo;Atoh1FRTcre;Ai93 (GCaMP6f) mutants to optically record the activity of Peri5 neurons in reduced brainstem spinal cord preparations and slices.
We have now produced the genotypes Phox2bFLPo;Atoh1FRTcre;Ai32 (ChR2) and Phox2bFLPo;Atoh1FRTcre;Ai35 (Archaerhodopsin-3).
- Anterograde tracing of Peri5 neurons
We have shown that Peri5 neurons selectively target hypoglossal and medial facial motoneurons, ispsilaterally. These motoneurons respectively innervate muscle for tongue protrusion, opening of the jaw, lowering of the lip and swallowing. Retrograde monosynaptic tracing from the diaphragm and from the masseter muscles indicate that Peri5 neurons are not premotor to either trigeminal or phrenic motor neurons.
-Anterograde tracing of RTN neurons
RTN terminals were identified in the Nucleus Tractus Solitarius (NTS), in the dorsal motor nucleus of the vagus (Xm) and in the preBötzinger complex (preBötC). Projections to pontine structures were also found in the vicinity of parabrachial/Kölliker-Fuse nucleus.
- Retrograde tracing from Peri5 neurons
We have attempted multiple strategies to achieve trans-synaptic retrograde labeling from Peri5 neurons. We have successfully obtained selective ?G-Rb infection of Peri5 neurons with two strategies, however the yield of transsynaptic labeled presynaptic neurons remains low whatever our efforts to optimize G-complementation. Interestingly, a comparable limitation applies to the RTN.
We will use digital holography to spatially restrict illumination to the Peri5 or the RTN.
We will directly evaluate the ventilation and the survival of mutants that have a combined and selective defect in the peri5 and the RTN ( Egr2::cre;R2::cre; Phox2b27AlaCKI) in normal and hypercapnic air.
We are currently mapping in greater details terminal projections of Peri5 neurons by using a AAV encoding the synapsin protein fused to GFP with a novel transgenic tool, not included in the original proposal.
-Additional strategy for anterograde tracing of Peri5 and RTN neurons
We have devised a novel method for anterograde labeling which does not require stereotaxic injections and could have more general uses. In brief, the construct allows combinatorial excisions of stop cassettes by a Cre-recombinase and a Flippase each driving expression of a given fluorophore addressed to a specific cellular domain. In flippase only-expressing cells recombination will result in cytoplasmic expression of Tomato (red labeling). Cre-recombinase only-expressing cells will not be labeled, while Flippase- AND Cre-expressing (intersection) cells will express a synapsin-GFP fusion protein (green labeling). Double XFLPo;YCre genotypes can now be used to visualize the green axonal terminals of X AND Y expressing neurons with the added value that green axonal terminals on X only expressing neurons will immediately be spotted as green boutons on red labeled cells. If X is chosen among genes that define a given cell type (e.g. Phox2b for visceral neurons) then this reporter may reveal that a Y subset of Phox2b neurons is connected to non-Y subsets of Phox2b neurons thus contributing to the demonstration that visceral circuits largely rely on Phox2b-Phox2b homotypic synapses. We are currently testing the efficiency of Cre and FLPo recombination using the Phox2bFLPo;Atoh1FRTcre line.
None for the moment
This project aims at deciphering the function of a genetically defined group of neurons in the mouse hindbrain, with cutting edge tools that are increasingly applied to the functional interrogation of neurons in the central nervous system. More specifically, it will target a cluster of neurons in the rostral hindbrain (or pons), presumably involved in the regulation of breathing. In the course of our previous studies on a rhythmogenic and chemoreceptive center of the hindbrain, the retrotrapezoid nucleus (RTN), we spotted a small population of neurons of unknown function and connectivity that share with the RTN a molecular signature —Phox2b+ and Atoh1+— and a type of location —surrounding a branchiomotor nucleus, in this case the trigeminal motor nucleus rather than the facial, and that we termed peri-trigeminal (Peri5) neurons. Several lines of evidence from our lab and from the literature, suggest that this structure could be involved in breathing or otherwise implicated in the lethality of newborn mice —and possibly of humans afflicted with congential central hypoventilation syndrome (CCHS)— when it is missing in combination with the RTN. This project is a concerted attempt at deciphering the function of this molecularly coded neuronal population. Using intersectional genetic tools in mice, we will monitor the activity of this nucleus by optogenetics, map its connectome by anterograde, retrograde and trans-synaptic viral tracing, delete it specifically with conditional mutations mediated by Cre and Flp recombinases, acutely interfere with its activity by chemogenetics in the newborn and the adult, and monitor the consequences on breathing, airway patency and orofacial rhythms. Finally we will take advantage of the unique molecular signature of the RTN and peri5 neurons to search for homologues in fish, to pave the way for an understanding of the evolution of respiratory rhythmogenesis.
Project coordination
Jean-François BRUNET (Institut de Biologie de l'Ecole Normale Supérieure)
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
Partner
CNRS Neuro-PSI
IBENS Institut de Biologie de l'Ecole Normale Supérieure
Help of the ANR 616,482 euros
Beginning and duration of the scientific project:
September 2015
- 42 Months
