SUPPLEMENTARY INFORMATION

Transcription

1 SUPPLEMENTARY INFORMATN doi:1.138/nture1276 prebötzinger units Respirtion Ammoni Inspirtion Expirtion 2 s 7 Fcil nucleus prebötzinger LRt 4 µm Figure S1. Trnsient exposure to mmoni inhiits ctivity in the prebötzinger complex in the nesthetized rt. A puff of mmoni suppresses rhythmic ursts in prebötzinger cells, which constitute the core of the inspirtory centrl pttern genertor for rething. The recording site in the prebötzinger is leled y deposit of the dye Chicgo sky lue. 1

2 RESEARCH SUPPLEMENTARY INFORMATN 12 Mesured inspirtion onset Breth numer 6-1 Intervening whisks Time from mesured inspirtion onset (s) Figure S2. Coordintion of whisking nd rething in one rt. Temporl reltionship etween whisking nd rething events. Rster plots of inspirtion nd protrction onset times reltive to ech reth re sorted y the durtion of the reth. In n individul niml, the intrinsic whisking oscilltion frequency is stle nd locked to the mesured rething onset time t oth sl respirtory frequencies s well s sniffing frequencies. During sl respirtion, the first, second, nd third whisks following inspirtory drive occur t stereotypic times reltive to rething. 2

3 SUPPLEMENTARY INFORMATN RESEARCH Pressure 2 Video Intervening whisks Odor Numer of reths c d 3 2 Bsl 25 Inspirtion onset Sniff 1 Protrction onset Coherence of whisking nd rething Breth numer Spectrl mplitude (Normlized) Whisking during sniffing Whisking during sl respirtion Sniff Bsl 1 2 ms Time 6 Hz 5 Hz Intervening whisks 1 Frequency (Hz) Time from inspirtion onset (s) 1. Figure S3. Coordintion of whisking nd rething in mice. () Experimentl procedures to mesure whisking nd rething. Hed-restrined mice were implnted with pressure trnsducer in the nsl cvity1, nd virisse were monitored with high speed videogrphy. () Simultneous mesurement of whisking nd rething. Viriss C2 position is in lue nd pressure is in red. Protrction nd inspirtion re upwrd. During sniffing, inspirtion is synchronous with viriss protrction on ech cycle (top). During sl respirtion, whisking is fster thn rething ut inspirtion remins synchronous with protrction (ottom). (c) Spectrl reltionship etween whisking nd rething. Histogrm of instntneous rething frequency for ech reth (top). Brething rtes ove 6 Hz re clssified s sniffing, nd elow 5 Hz re clssified s sl respirtion. Whisking spectr re plotted for periods of sniffing (red) nd periods of sl respirtion (lck). Coherence etween whisking nd rething for periods of sniffing nd periods of sl respirtion. Dshed lines represent thresholds for sttisticl significnce (p <.5). (d) Temporl reltionship etween whisking nd rething events. Rster plots of inspirtion nd protrction onset times reltive to ech reth re sorted y the durtion of the reth, s in Figure 1. At high respirtory rtes, whisking nd rething show 1:1 reltionship, ut t lower frequencies there re intervening whisks etween reths. These dt refute the clims of Co, Roy, Schdev nd Heck (212, Dynmic correltion etween whisking nd rething rhythms in mice. J Neurosci 32, ). W W W. N A T U R E. C O M / N A T U R E 3

4 RESEARCH SUPPLEMENTARY INFORMATN Phse response of whisking to rething Whisks Onset of protrction T T 1 PDF c Chnge in phse of whisk onset, ΔΦ whisk.1 - Phse in whisk cycle,φ whisk Protrction * t reset Inspirtion, Φ reset Retrction 2 Phse of reth within whisk cycle, Φ reset Next whisk delyed Next whisk dvnced Figure S4. Phse resetting of whisking y rething () Schemtic descriing the phse-response of whisking reltive to the onset of inspirtion. For ech whisk, the time t which inspirtion occurs fter the onset of protrction is defined s t reset ; in this nlysis inspirtion plys the role of n externl perturtion. The expected durtion of the whisk is denoted y T nd, s the whisking frequency is stle throughout out, is tken s the durtion of the preceding whisk. The durtion of the pertured whisk is denoted y T 1. These times re normlized s the phse of the reth within the whisk cycle, defined y φ reset = 2π(t reset /T ) long with the resultnt chnge in whisking phse, defined y φ whisk = 2π(T 1 -T ) / T 1. () Proility density of φ reset t during sl respirtion. Inspirtion occurs t ll phses of the whisking rhythm during sl respirtion. (c) Phse response of whisking to the onset of inspirtory drive during sl respirtion. Onset times re djusted s shown in Figure 1d. Symols re defined ove. The red sterisk represents the clculted perturtion for the exmple in the time-line in pnel. 4

5 SUPPLEMENTARY INFORMATN RESEARCH Shift in whisking period y rething T1 T (ms) 2 Inspirtion locked whisks 1 1 Intervening whisks shortened y reth Tr: Time of reth within whisk cycle (ms) Shift in rething period y whisking T1 T (ms) Brething period unffected y whisking Tr: Time of whisk within reth cycle (ms) Figure S5. Response of the whisking rhythm to rething versus the rething rhythm to whisking To emphsize the symmetric interction etween whisking nd rething, we plot the shift in period (T1 -T) of ech ehvior s function of the time within the cycle when the other ehvior occurs (treset). As in Figure S4, T represents the expected period of the pertured ehvior, tken s the period of the cycle prior to tht which contins n interrupting event, nd T1 represents the interrupted period. Unlike Figure S4, oth sniffing nd sl respirtory frequencies re included in the nlysis to permit symmetric comprison etween the response of whisking to rething nd the response of rething to whisking. () Shift in whisking period (T1 -T) reltive to the reset y n inspirtion (t times treset). As in Figure S4, there is is for the intervening whisks to e shortened y the onset of rething during sl respirtion. The inhltion locked whisks during sniffing re not in stedy stte nd not so constrined. () Shift in respirtory period (T1 -T) reltive to the reset y viriss protrction (t times t reset ). There is no pprent is for the period of rething to e shifted y the presence of whisk during either sniffing or sl respirtion. 5

6 RESEARCH SUPPLEMENTARY INFORMATN Inspirtory-locked whisks Intervening whisks Intrinsic EMG 5 uv Extrinsic EMG 5 uv Time from protrction onset (s) Figure S6. Cross-correltion etween EMG ctivity nd viriss movement. Cross-correltion of the smoothed EMG for intrinsic (green) nd extrinsic (lck) muscles with the protrction onset times for inspirtory nd intervening whisks. Solid trces represent correltion with inspirtory whisks nd dshed trces represent correltion with intervening whisks. Bnds represent 95 % confidence intervls. 6

7 SUPPLEMENTARY INFORMATN RESEARCH Dorsl Rostrl.2 mv 3º LRt 1s 1 mm Rostrl Dorsl LRt c Rostrl Dorsl LRt d Rostrl Dorsl LRt e Lterl Dorsl Am Figure S7. Compendium of spiking records from ll virt whisking units during rething nd whisking in lert rts. (-e) Concurrent recordings of rething (red), whisking (lue), nd multiunit spike ctivity (lck) in the IRt for ll whisking units. The loction of the recording site leled with Chicgo sky lue is shown on the right sgittl section (pnels to d) or coronl section (pnel e) counterstined with neutrl red (pnels, c, nd e) or cytochrome oxidse (pnels nd d). Lndmrks, including the fcil nucleus (), inferior olive (), lterl reticulr nucleus (LRt), nd miguus nucleus (Am), re outlined in lck. Arrowheds mrk the leled recording site. Scle rs re s defined in pnel. W W W. N A T U R E. C O M / N A T U R E 7

8 RESEARCH SUPPLEMENTARY INFORMATN Injection site LRt 1 mm 1 µm Figure S8. Kinic cid injection site nd surrounding rinstem tissue. Injection t this site resulted in sustined, coordinted ipsilterl whisking. Whisking ppered norml the dy following the injection nd therefter (dt not shown). () The injection site is leled with iotinylted dextrn mine, nd the tissue is counterstined with n ntiody ginst neuronl nucler protein fter three dys of recovery. () Mgnified view of the white ox in pnel. Cells round the injection site remin intct.. 8

9 SUPPLEMENTARY INFORMATN RESEARCH Iplterl D1 2º Viriss position Ipsilterl D2 Ipsilterl D3 Contrlterl D2 1 s Time Figure S9. Coordinted movement of ipsilterl virisse following kinic cid injection in the vcinity of the virt Simultneously recorded motion of three neighoring virisse, i.e., D1, D2 nd D3, on the ipsilterl side, nd the D2 viriss on the contrlterl side, fter ctivtion of whisking y kinic cid injection. 9

10 RESEARCH SUPPLEMENTARY INFORMATN 7 minutes fter kinic cid injection 2 Bsl 3 Hz Breth numer 1 9 minutes fter kinic cid injection 3 Breth numer 15 Bsl 3 Hz Time from mesured inspirtion onset (s) Figure S1. Lck of coordintion of kinic cid-induced whisking nd rething. Temporl reltionship etween whisking nd rething events for kinic cid induced whisking in the lightly nesthetized rt. Rster plots of inspirtion (red dots) nd protrction (lue dots) onset times reltive to ech reth re sorted y the durtion of the reth. () At sl respirtory frequencies, correltion with event times ws low nd significnt in only one of eleven different twenty minute epochs (p <.1) cross three nimls; shown here is this single cse. () The correltion of the remining ten out of the eleven epochs ws found to esttisticlly insignificnt (p >.5); one of the ten cses is shown here. 1

11 SUPPLEMENTARY INFORMATN RESEARCH Electrolytic lesion Iotenic cid lesion c Sindis virus lesion Whisking mplitude (º) 2 2 Ipsilterl side Contrlterl side Ipsilterl side Contrlterl side Ipsilterl side Contrlterl side 1 s 8 Ipsilterl mplitude (º) Contrlterl mplitude (º) Contrlterl mplitude (º) Contrlterl mplitude (º) SpVI Amiguus Amiguus 1 mm Figure S11. Three different methods of lesion in the virt tht result in ipsilterl whisking impirment () Exmple of whisking out following n electrolytic lesion in the virt (top). As in Figure 5, full nlysis of ipsilterl versus contrlterl whisk mplitudes revels the functionl completeness of the lesion (middle; ech dot represents one whisk, circle represents the men, nd lines represent the inter-qurtile rnge). Histologicl nlysis confirms tht the lesion is in the virt; coronl section stined with neutrl red (ottom). This pnel is repliction of Figure 5, included here for comprison to other lesion methods. () Exmple of whisking out fter n excitotoxic lesion in the virt mde y pressure injection of iotenic cid (top). The nlysis of whisking mplitudes (middle) is s in pnel. The histologicl section (ottom) shows the extent of neuronl degenertion fter stining with α-neun. (c) Exmple of whisking out fter lesion in the virt mde y microinjection of Sindis virl vector tht codes for the expression of green-fluorescent protein (top). The nlysis of whisking mplitudes (middle) is s in pnels nd, nd the histology (ottom) is s in pnel. We further show n djcent 3 µm section stined with Luxol fst lue to lel myelin nd neutrl red to lel Nissl odies; the outlined region corresponds to the loction where there ws loss of cells s reveled y the α-neun stining. 11

12 RESEARCH SUPPLEMENTARY INFORMATN.5 mm Fluorogold Rostrl Fcil nucleus Lterl reticulr nucleus Inferior olive 1 mm.5 mm Neuroiotin Rostrl Fcil nucleus Lterl reticulr nucleus c d Inferior olive 1 mm.2 mm.2 mm Figure S12. Fluoro-Gold nd Neuroiotin retrogrdely lel virt cells tht project to the fcil motor nucleus. () Cells re retrogrdely leled in virt nd PCRt following n injection of Fluoro-Gold in the lterl spect of the fcil nucleus (inset). Sgittl section. () A similr pttern of leling is oserved fter injection of Neuroiotin into the lterl fcil nucleus (inset). (c-d) Mgnifiction of the virt regions outlined in pnels nd, respectively. 12

13 SUPPLEMENTARY INFORMATN RESEARCH 1 mm c LRt 1 mm Figure S13. Fluoro-Gold retrogrde leling for chrcteriztion of the neurotrnsmitter phenotypes of fcil pre-motoneurons. () Exmple of Fluoro-Gold injection site in the lterl fcil nucleus. () Region in which counts of cells doule-leled with Fluoro-Gold nd in situ hyridiztion for vglut2, GlyT2, or GAD67 were performed. (c) Mgnified view of the cell counting region in pnel. 13

14 RESEARCH SUPPLEMENTARY INFORMATN Retrogrde lel from fcil nucleus In situ hydridiztion proe in virt Overlp shows doule leled somt Glycine (GlyT2 proe) Glutmte (VGluT2 proe) d 1 d 2 γ-minoutyric cid (GAD proe) d 3 5 µm Figure S14. Determintion of the neurotrnsmitter phenotype of neurons tht project from the virt to the fcil motoneurons tht drive protrction of the virisse. Neurons were leled vi retrogrde trnsport of Fluor-Gold from the lterl spect of the fcil nucleus (left column) nd the tissue ws processed for in situ hyridiztion (middle column) with proes for glutmte (top row), glycine (middle) nd γ-minoutyric cid (ottom). The merged imges (right colum) revel doule-leled cells, denoted with rrows. 14