neuronal network oscillations and their interactions are thought to be important for perception , sensory - motor coordination , and memory processes .
according to a recent hypothesis , the breathing rhythm serves as a clock for binding several orofacial senses to a common percept . in line with this hypothesis ,
it was found that whisking synchronizes with breathing [ 5 , 6 ] and that slow oscillations in the whisker barrel cortex of waking mice are locked to respiration .
recently , we demonstrated in the dentate gyrus of anaesthetized   and awake   mice a respiration - related rhythm ( rr ) clearly distinct from theta oscillations .
discrimination of the rr from other slow rhythms requires registration of respiration , preferably as an electrical field potential which can be easily recorded together with other electrophysiological data .
intranasal pressure sensors or thermocouples implanted into the nasal cavity [ 1012 ] are commonly used in rodents but are invasive devices which may compromise natural behavior . as a less invasive alternative , whole - body plethysmography [ 1315 ] provides a reliable , state - independent biomechanical measure of respiration .
however , plethysmography requires housing of the animals in specialized and closed small chambers , making it unpractical for use in combination with tethered brain recordings or complex behavioral tasks . for decades
, it had been known that local field potentials ( lfp ) of the olfactory bulb ( ob ) of mammals are entrained by nasal respiration [ 1619 ] .
this signal has already been used to measure respiration rate in freely moving rats . however ,
the reliability of ob lfp for estimating breathing and sniffing frequencies in mice has not been systematically studied and verified by alternative approaches , especially not through different states of vigilance . in the present study ,
we simultaneously used plethysmography , nasal thermocouple , and lfp recordings in ob during various sleep - wake states of mice .
all experiments were performed according to the guidelines of the european science foundation   and the us national institutes of health guide for the care and use of laboratory animals ( institute of laboratory animal research ) and were approved by the governmental supervisory panel on animal experiments of baden wrttemberg at karlsruhe ( g84/13 ) .
all efforts were made to minimize animal suffering and to reduce the number of animals used .
c57bl/6n mice were purchased at 70 or 84  d of age from charles river ( sulzfeld , germany ) .
animals were housed in groups of four inside a ventilated scantainer ( scanbur , dk ) on an inverted 12  h/12  h light / dark cycle ( light on 8:00 pm ) for a minimum of 2 weeks .
mice had free access to food and water . after chronic electrode implantation , mice were kept individually throughout the experiment .
a total of 17 c57bl/6n mice ( 16 female and one male ; 1329 weeks old ; weight : 2227  g ) were anesthetized with isoflurane in medical oxygen ( 4% isoflurane for induction , 1.5%2.5% for maintenance , flow rate : 1  l per minute ) . for analgesia , 0.1  mg / kg of buprenorphine
was injected subcutaneously before and 8  h after surgery ( for details see ) .
after exposure of the skull , holes of 0.51.0  mm in diameter were drilled above the ob , the parietal cortex , and cerebellum according to stereotaxic coordinates ; see below for coordinates based on .
two stainless steel watch screws ( 1  3  mm ) over the cerebellum served as ground and reference electrode . for monitoring the temperature of nasal air flow , precision fine
bare wire thermocouples ( 80  m diameter , omega engineering , part # 5tc - tt - ki-40 - 1  m ) were implanted into both nasal cavities ( 11  mm anteroposterior ( ap ) , 0.5  mm mediolateral ( ml ) ) .
a pair of varnish - insulated tungsten wires ( 50  m ) was implanted into the granule cell layer of left ob ( ap : + 4.5 ; ml : 0.8 ; 1.3  mm ventral ) . for restraining the head of the animal , a steel pin ( 2.1  mm diameter , 14  mm length )
one week after surgery , experiments began with a 1  h recording session in the plethysmograph ( see below ) or in a custom - built head - fixed setup with a treadmill for voluntary running .
extracellular signals were filtered ( 1500  hz ) , amplified ( rha2000 , intan technologies , los angeles , usa ) , digitized ( 2.5  khz ) , and stored for offline analysis .
a three - dimensional accelerometer mounted on the amplifier board at the animals head allowed movement detection .
mice habituated quickly to the head - restraining conditions and tolerated it well for up to 1  h. the whole - body plethysmograph ( pg ) consisted of a transparent cylindrical box ( 78  mm inner diameter , 165  mm height ) which was connected to a reference chamber and adapted for collection of lfp ( emka technologies , sas , france ) .
classification of vigilance states was based on ( 1 ) the level of accelerometer activity ( figure 1 : mov ; waking ( wk ) > nrem , rem ) ; ( 2 ) the amount of high amplitude slow wave activity in the neocortex ( figure 1 : parietal cortex ( pac ) , slow waves ; nrem > wk , rem ) ; ( 3 ) the amount of regular theta oscillations in the parietal cortex overlaying the dorsal hippocampus ( figure 1 : pac , theta ; rem > nrem , active wk > nrem ) . for detailed description of behavioral staging ,
natick , ma ) using built - in and custom - written routines ( for more details see ) .
power spectral density was calculated by means of the welch periodogram method using the pwelch.m function from the signal processing toolbox ( 50% overlapping , 4  s hamming windows ) .
time - frequency power decomposition was obtained by means of the spectrogram.m function ; sliding windows of 2  s and 50  ms time steps were used ; phase coherence was obtained by means of the mscohere.m function from the signal processing toolbox , using 2  s windows with 50% overlap .
for all types of data analysis , lfp signals of 25  s duration were used .
after the experiments , animals were deeply anesthetized with isoflurane and perfused transcardially with pbs and subsequently with 4% paraformaldehyde ( pfa ) .
brains were carefully dissected and stored in pfa overnight , and coronal sections were cut ( 50  m ) , mounted , and stained with cresyl violet .
data are expressed as mean  sem . for group comparisons of normally distributed data ( kolmogorov - smirnov test ) , we used t - test or repeated measures anova followed by tukey 's multiple comparison test . for data with non - gaussian distribution , we used the nonparametric friedman test , mann - whitney test , or the wilcoxon signed rank test .
respiration was recorded using a thermocouple ( tc ) chronically implanted into the nasal cavity .
simultaneously , thoracic movements were detected with whole - body plethysmography ( pg ) indicating relative values of respiratory air flow ( ml / s ) .
in addition , local field potentials ( lfp ) from the granular cell layer of the olfactory bulb ( ob ) showed fluctuations entrained by nasal respiration as described earlier .  
figure 2 illustrates similarity of the three methods in a representative example of waking immobility ( figure 2(a ) : raw signals and figure 2(b ) : time - frequency distribution ) .
as expected , power spectral density analysis results in power peaks of identical frequency ( 4.42  hz ; figure 2(c ) ) .
phase coherence between pg and ob ( figure 2(c ) , lower graph , red ) or tc and ob ( figure 2(c ) , lower graph , blue ) was largest ( ~0.9 ) at the frequency of respiration with its harmonic at 8.84  hz .
we next investigated how peak power of the rr in ob varied with changing breathing frequency .
respiration rate in waking mice varies strongly , especially during interspersed spouts of sniffing [ 9 , 11 ] .  
figure 3 shows a representative example of an abrupt transition from slow breathing to fast sniffing . at high sniffing rates , the amplitude of the pg signal increased while the amplitude of the rr in ob decreased ( figure 3(a ) , raw signals ) . despite
the amplitude differences similar prominent rhythms were found for all three signals in the spectrograms ( figure 3(b ) ) and in power spectral density ( psd ) plots , at low ( figure 3(c ) , black ) and high frequencies ( figure 3(c ) , blue ) . for quantitative analysis , periods of waking were sorted into frequency bins of breathing / sniffing obtained by pg ( figure 3(d ) ) .
psds of the three simultaneously recorded respiration signals and coherence between pg and ob ( not shown ) were calculated for each interval .
peak powers of pg , tc , and rr in ob were z - scored and averaged .
pg peak power increased with respiration frequency ( figure 3(d ) : red ; n = 8 ; p < 0.0005 ; friedman test ) , whereas tc power did not change ( figure 3(d ) : gray ; n = 8 , p = 0.22 ; repeated measures anova ) .
in contrast , peak power of the rr in ob decreased in the highest frequency bin ( 8 to 10  hz ) compared to the three remaining bins ( figure 2(d ) : black ; n = 8 ; p < 0.01 ; repeated measures anova ; post hoc tukey test ) . mean coherence between pg and ob lfp did not change ( not shown ; n = 8 ; p = 0.15 ; repeated measures anova ) .
this indicates high coherence between ob , tc , and pg for all respiratory frequencies during waking . to reduce respiratory variability , we next performed head - fixed experiments where the behavior of the mouse was basically clamped to either immobility or running on a circular treadmill and minimized sniffing
. only periods of running were used here and divided into 1  hz bins of increasing breathing frequency obtained by simultaneously recorded tc .   figure
3(e ) displays z - scored averages of rr peak power in ob ( n = 11 mice ) which linearly decreased with increasing respiration frequency ( n = 7 ; r = 0.958 ; p < 0.001 , pearson 's correlation ) .
large amplitude slow waves in the delta frequency range are characteristic features of nonrapid eye movement ( nrem ) sleep .
delta waves are superimposed on local field potentials in ob hiding traces of respiration - related fluctuations between 1.2 and 3.5  hz which could be reliably detected with pg ( see nrem in figure 4(a ) , raw traces ) .
respiration - related signals in tc disappeared during nrem ( figures 4(a ) and 4(b ) ) , whereas the signal in pg remained .
in contrast to the clear power peak in pg ( figure 4(c ) , upper panel ) , no distinctive peaks were discernible in spectrograms from tc and ob signals , respectively ( figure 4(c ) , middle and lower panel ) .
both rr in ob and respiratory signal in tc regularly returned during microarousals ( see asterisks in figure 4(b ) ) . during parts of rem sleep , especially at low breathing frequencies ,
rr in ob recovered ( figure 5(a ) , low ; figure 5(b ) ) , whereas the tc signal remained absent .
clear power peaks were found for pg and ob for both low ( figure 5(c ) , upper and lower panel , black ) and high ( figure 5(c ) , blue ) breathing frequencies , in contrast to tc where no corresponding peaks were discernible in the power spectral density graph ( figure 5(c ) , middle panel ) . during rem sleep ,
rr in ob was detectable , but the signal was less reliable as compared to awake animals , especially at high breathing frequencies ( figures 5(a ) and 5(c ) , lower panels , blue ) . for all corresponding bins of breathing frequency , rr peak power was smaller during rem compared to waking ( 1.25 to 3.25  hz : n = 6 ; p < 0.05 ; t - test ; 3.25 to 5.25  hz : n = 6 ; p < 0.01 ; t - test ; 5.25 to 7.25  hz : n = 6 ; p < 0.05 ; wilcoxon test ) .
figure 5(d ) shows peak power averages of six animals of pg ( red ) and ob ( black ) recorded during rem sleep binned into three breathing frequency ranges as above .
pg peak power did not depend on breathing frequency , in contrast to rr peak power in ob ( figure 5(d ) ; black ; n = 6 ; p < 0.01 ; friedman 's test ) . likewise , coherence between pg and ob signals decreased between the lowest and highest breathing frequency bins ( figure 5(e ) ; n = 6 ; p < 0.01 ; friedman 's test ) . coherence between pg and ob was also lower in rem compared to waking ( not shown ) for the two highest frequency bins ( n = 6 ; p < 0.05 and p < 0.01 ; wilcoxon test ) . in summary , plethysmographic data provided the only reliable measure of respiration throughout nrem sleep and was more reliable during rem sleep at high breathing frequencies compared to ob lfp which was a good indicator of respiration during rem sleep with low respiration rates . 

figure 6 illustrates representative examples of coherence and its harmonics between all three signals : pg - ob ( red ) , tc - ob ( blue ) , and pg - tc ( green ) at different ranges of respiration during waking ( a ) , nrem sleep ( b ) , and rem sleep ( c ) . in waking ( figure 6(a ) ) , large peaks of coherence between all signals correspond to respiration frequency ( pg psd : black ) indicating the reliability of all three signals to measure respiration . during waking and rem sleep ,
there is no delta activity in ob ; therefore , the lfp is a good indicator of respiration and its peak in psd corresponds to the respiration rate .
however , in nrem sleep , peaks at respiration rate remained only for coherence between pg and ob .
coherence analysis indeed demonstrates that the respiration - related rhythm is preserved in ob during sleep .
however , for coherence analysis , a second source of respiration is required during nrem sleep ; the ob lfp alone is not sufficient as unequivocal signal of respiration due to multiple equivocal peaks in the power spectral density of the ob lfp .
in the present study , we report slow local field potentials ( lfp ) in the granular cell layer of olfactory bulb ( ob ) which reliably indicate respiration in waking mice .
this was verified by simultaneous measurement of nasal thermocouple ( tc ) signals and plethysmography ( pg ) .
our results confirm reports from several decades ago which described slow rhythmic lfps in the ob related to nasal air flow and breathing [ 1618 ] . at the time
, however , these data could not be independently verified due to lack of adequate digital analysis tools . during subsequent years
, the concept of respiration - related rhythmic fluctuations of lfps in ob was progressively replaced by a terminology which subsumed slow ob rhythms as olfaction - related theta oscillations [ 2528 ] ; see also discussion in . from a different viewpoint ,
however , theta oscillations are not just rhythmic fluctuations in the respective frequency range ( 412  hz ) but share the characteristic behavioral features of hippocampal theta rhythms .
theta oscillations occur during locomotion , exploration , arousal , or fear [ 30 , 31 ] or in rem sleep .
in contrast , respiration - entrained rhythms in ob , if not masked by delta waves during sleep , are present in any behavioral state .
they reflect respiration , and their amplitude is largest during relaxed wakefulness [ 9 , 33 ] , a state when theta rhythm is absent [ 29 , 34 ] .
indeed , we have recently demonstrated that a clearly distinct respiration - related rhythm ( rr ) occurs alone or simultaneously with theta oscillations both in ob and in upstream regions like the dentate gyrus of the hippocampal formation [ 8 , 9 ] .
for example , tracheotomy abolishes rr but not theta oscillations , whereas atropine diminishes theta but does not affect rr . in the present study , we report that only pg , but not tc or ob lfps , is trustworthy proxy for respiration during nrem sleep .
while rr in ob repeatedly returned during nrem and especially during rem sleep , the tc signal was absent throughout nrem and rem sleep .
this may be in part explained by a decrease in nasal air flow during these states .
indeed , total inspiratory flow in mice decreases continuously from quiet waking to nrem and rem sleep .
other factors , like redirection of air flow inside the nasal cavity , may contribute to the disappearance of respiration signals at the tc .
interestingly , both signals , tc and rr in ob , regularly reappeared during microarousals , providing a potential indicator of these brief states with enhanced vigilance . in summary , investigating respiration and its related oscillations in the mouse brain during nrem sleep requires pg or other signals different from tc and rr in ob . in waking states of freely moving and behaving mice and in parts of rem sleep with low respiration rates ,
however , ob lfps can be reliably used to measure respiration . in addition , we report that rr peak power decreases with increasing respiration rate under several behavioral conditions in wakefulness and rem sleep .
our findings from mice are in good agreement with earlier reports on anesthetized rats [ 37 , 38 ] . in any case , measuring respiratory rhythm during nrem sleep and separating it from delta activity require great caution .
local field potentials in the olfactory bulb of waking but not sleeping mice reliably reflect respiration .
this holds also true for nasal thermocouples . in sleeping mice , the most accurate method to record breathing is whole - body plethysmography .
local field potentials from olfactory bulb are reliable in parts of rem sleep with low respiration rates but alone are not sufficient in nrem sleep .
 it is well established that local field potentials ( lfp ) in the rodent olfactory bulb ( ob ) follow respiration . this respiration - related rhythm ( rr ) in ob depends on nasal air flow , indicating that it is conveyed by sensory inputs from the nasal epithelium . 
 recently rr was found outside the olfactory system , suggesting that it plays a role in organizing distributed network activity . 
 it is therefore important to measure rr and to delineate it from endogenous electrical rhythms like theta which cover similar frequency bands in small rodents . in order to validate such measurements in freely behaving mice , we compared rhythmic lfp in the ob with two respiration - related biophysical parameters : whole - body plethysmography ( pg ) and nasal temperature ( thermocouple ; tc ) . during waking , 
 all three signals reflected respiration with similar reliability . 
 peak power of rr in ob decreased with increasing respiration rate whereas power of pg increased . during nrem sleep , respiration - related tc signals disappeared and large amplitude slow waves frequently concealed rr in ob . in this situation , pg provided a reliable signal while breathing - related rhythms in tc and ob returned only during microarousals . in summary , local field potentials in the olfactory bulb do reliably reflect respiratory rhythm during wakefulness and rem sleep but not during nrem sleep . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions