Organization of the core respiratory network: Insights from optogenetic and modeling studies
[['Rhythmic movements such as breathing and locomotion are produced in the nervous system by central pattern generator ( CPG ) networks consisting of interacting inhibitory and excitatory circuits that constitute the basic neural machinery generating motor behavior .', 'Defining the structural-functional arrangement of CPG circuits remains a central problem in many neural motor control systems .', 'In the present studies , we have addressed this problem of delineating the configuration of core circuits of the mammalian brainstem respiratory CPG .', 'The basic pattern of respiratory activity includes three major phases: inspiration , post-inspiration , and late expiration [1 , 2] .', 'This pattern is generated within bilaterally located medullary ventral respiratory columns ( VRC ) , each of which includes two key compartments , the pre-Bötzinger ( pre-BötC ) and Bötzinger ( BötC ) complexes , postulated to represent the core of the respiratory CPG [3–11] .', 'The pre-BötC contains a heterogeneous population of excitatory neurons , including cells with intrinsic bursting properties , with mutual synaptic interconnections that generate rhythmic bursts of inspiratory activity and drive inspiratory motor output [12–17] .', 'This structure also contains circuits of GABAergic and glycinergic inhibitory neurons [18–21] , which are active during the inspiratory phase and provide phasic inhibition that is widely projected to other brainstem compartments , including to BötC circuits to inhibit their expiratory neurons .', 'The BötC mostly contains post-inspiratory and augmenting expiratory neurons that provide post-inspiratory and expiratory inhibition [22–25] .', 'These neurons inhibit pre-BötC inspiratory neurons during expiration to coordinate expiratory and inspiratory phases of breathing .', 'Although post-inspiratory neurons are predominantly located in the BötC , they are widely distributed within the VRC and present in other brainstem compartments including the pre-BötC [9 , 26 , 27] .', 'The current experimental data suggest that generation and shaping of the basic respiratory pattern are based on the intrinsic bursting properties of , and interactions between , the excitatory neurons within the pre-BötC and involve inhibitory interactions between different neuron populations within and between the pre-BötC and BötC regions .', 'The pre-BötC/BötC core circuitry is proposed to interact with other brainstem compartments , including several pontine structures [3 , 28–30] , the retrotrapezoid nucleus/parafacial respiratory group ( RTN/pFRG; [31–36] , and the nucleus tractus solitarii ( NTS , mediating multiple inputs and feedbacks to the respiratory network ) , which modulate and adjust operation of the core respiratory network and the pattern of generated respiratory activity to the current physiological and metabolic conditions [37–39] .', 'The basic connectome of interactions among respiratory neuron populations in the pre-BötC and BötC compartments has been proposed in a series of computational models [3–8 , 10 , 11 , 40] .', 'These models followed the key conceptual suggestions that ( 1 ) although the pre-BötC is capable of autonomous generation of rhythmic bursting when isolated in vitro , in more intact preparations and in vivo this structure is embedded in a larger respiratory network where its operation is modulated by interactions with other brainstem compartments , and particularly depends on mutual inhibitory interactions with the BötC , so that both the pre-BötC and BötC are critically involved in the generation and shaping of the normal ( eupneic ) three-phase rhythmic respiratory pattern , and ( 2 ) inhibitory interactions between and within these core compartments are fundamentally involved in generating the normal rhythmic respiratory neural activity .', 'Accordingly , disruption of these interactions may cause a switch from the eupneic neural pattern of breathing defined by network interactions to either an intrinsic rhythmic activity originating within the pre-BötC , or to apnea or an abnormal apneustic activity .', 'The above concept and the related computational models were indirectly supported by multiple experimental data [2 , 41–44] .', 'However , this concept and , specifically , the critical role of inhibitory interactions in respiratory rhythm generation were challenged by a study from Janczewski et al . [45] , in which fast inhibitory neurotransmission was pharmacologically suppressed in anesthetized rats in vivo .', 'Although these authors acknowledged the important role of inhibition in “shaping the pattern of respiratory motor output , assuring its stability , and in mediating reflex or volitional apnea” , they concluded that: ( 1 ) “postsynaptic inhibition within the pre-BötC and BötC is not essential for generation of normal respiratory rhythm in intact mammals” [45] , and ( 2 ) “bilateral ablation of BötC did not result in apnea” and did not change respiratory frequency and phase durations [45] and hence the BötC does not play a role in respiratory rhythm generation during normal eupneic breathing .', 'Subsequently Sherman et al . [46] , who optogenetically targeted glycinergic pre-BötC neurons in mice to investigate their role in respiratory rhythm generation , similarly concluded that local inhibitory neurons are not essential for rhythm generation .', 'However , the latter study was limited to optogenetic manipulation of only glycinergic neurons in the pre-BötC and did not investigate the potential role of the BötC and its interactions with the pre-BötC .', 'In contrast , another recent study [47] showed that', '( a ) optogenetic activation of BötC neurons resulted in depression of the respiratory rhythm , and', '( b ) the effects of transient optogenetic stimulation of pre-BötC neurons depended on the phase of the respiratory cycle at which it was applied .', 'Specifically , there was an insensitive post-inspiratory period during which the next inspiratory burst could not be triggered by optogenetic stimulation of the pre-BötC [47] .', 'This insensitive period could result from a post-inspiratory inhibition originating in the BötC or within the pre-BötC .', 'The results of these studies are consistent with the original concept of an important contribution of BötC inhibitory circuits and their interactions with pre-BötC circuits in respiratory rhythm and pattern generation .', 'Resolving these issues requires systematic investigations using site-specific targeting of inhibitory neurons in the pre-BötC and BötC .', 'In the present study , we thus employed an optogenetic strategy with a transgenic mouse line expressing Cre recombinase controlled by the vesicular GABA transporter ( VGAT ) promoter to express Channelrhodopsin-2 ( ChR2 ) in both GABAergic and glycinergic inhibitory neurons .', 'Our experiments were performed with in situ perfused brainstem-spinal cord preparations from adult transgenic mice that readily enable site-specific laser stimulation of inhibitory neurons within the pre-BötC and BötC regions during electrophysiological recording for analysis of perturbations of circuit activity and motor output .', 'Single laser pulses of various intensities delivered at different phases of the respiratory cycle as well as series of pulses or sustained laser stimulations were applied either to the pre-BötC or the BötC regions .', 'The photo-induced perturbations were regionally-specific and were dependent on stimulus intensity , duration , and phase of photostimulation in the respiratory cycle .', 'The results of these studies were consistent with our original concept confirming an important role of the BötC and of inhibitory circuit interactions between the pre-BötC and the BötC for operation of the respiratory CPG .', 'To further examine and explain the results obtained in our study we revised our previous computational model of pre-BötC and BötC microcircuits by incorporating an additional population of post-inspiratory inhibitory neurons ( post-I ) in the pre-BötC which was based on the knowledge that these neurons are widely distributed in the VRC , including the pre-BötC [1 , 2 , 9 , 27 , 48] .', 'The revised model was able to reproduce our experimental perturbations of circuit activity with sustained and phase-dependent short-duration activation of inhibitory neurons in the pre-BötC and BötC .', 'The model was then further tested by its ability to reproduce previously published data showing the effects of site-specific photostimulation of neurons in the pre-BötC and BötC regions [47] , which were reproduced without any additional adjustments to the model parameters .', 'The circuit configuration proposed in the model leads to several testable predictions about respiratory network organization and the specific roles of different brainstem neuron populations and how their interactions contribute to generating and shaping the neural breathing pattern .'], ['We histologically validated the VGAT-Cre driver mouse line by verifying , via confocal fluorescence microscopy in fixed serial sections , Cre-driven tdTomato labeling of neurons in the pre-BötC and BötC ( Fig', '1 ) in the double Tg line VGAT-tdTomato ( n = 5 ) , and by confirming co-labeling of glycine antibody ( Fig 2A ) or GABA antibody ( Fig 2B ) in tdTomato-expressing neurons within the pre-BötC and BötC in the VGAT-tdTomato line ( n = 2 each ) .', 'We also confirmed tdTomato labeling of neurons distributed extensively throughout the medullary reticular formation , including in ventrolateral medullary respiratory regions caudal and adjacent to the pre-BötC ( e . g . , in the rostral ventrolateral respiratory group ( rVRG ) ( Fig 1 ) .', 'We subsequently verified Cre-driven ChR2-EYFP expression in VGAT-positive tdTomato labeled neurons within the pre-BötC and BötC regions in the triple Tg line VGAT-tdTomato-ChR2-EYFP ( n = 3 ) by two-photon microscopy in “live” neonatal medullary slices in vitro .', 'The ChR2-EYFP fusion protein was heavily expressed in processes and somal membranes of VGAT-tdTomato labeled neurons within the pre-BötC ( Fig 2C ) and BötC .', 'To verify the efficacy of photostimulation ( 473 nm , 0 . 5–5 mW ) of ChR2-expressing pre-BötC inspiratory VGAT-positive neurons , we performed combined optogenetic stimulation and whole-cell patch-clamp recordings in rhythmically active in vitro neonatal medullary slice preparations from the VGAT-tdTomato-ChR2-EYFP mouse lines ( n = 3 ) .', 'These slices effectively isolate the bilateral pre-BötC along with hypoglossal ( XII ) motoneurons to monitor inspiratory XII activity [17] , allowing whole-cell recordings from rhythmically active inspiratory pre-BötC neurons and simultaneous laser illumination to the recorded neuron ( Fig 3A ) .', 'We performed current-clamp recording from tdTomato-labeled neurons , in which co-expression of ChR2 was confirmed with two-photon single optical plane live images ( Fig 3B ) , and identified rhythmically active pre-BötC inspiratory neurons , which were active in phase with integrated inspiratory XII nerve activity ( ∫XII ) .', 'The membrane potential of these functionally identified VGAT-positive pre-BötC inspiratory neurons was depolarized by 10 . 0 ± 1 . 0 mV at 5 mW laser power ( n = 8 neurons ) with fast kinetics occurring within 12 ms , and recovery within 25 ms after terminating laser illumination ( Fig 3C ) .', 'Summary data ( n = 8 neurons from 3 slice preparations , mean ± SEM ) shown in Fig 3D illustrates that ChR2-mediated depolarization of VGAT-positive inspiratory pre-BötC neurons was laser power-dependent , and importantly demonstrates that laser illumination caused membrane depolarization of VGAT-positive ( inhibitory ) inspiratory neurons in all cases with the applied laser power ranging from 0 . 5 to 5 mW .', 'The observed membrane depolarization of these neurons could also include inhibitory effects on these neurons due to photostimulation of ChR2-expressing terminals from other VGAT-positive neurons with somas located outside of the pre-BötC providing inhibitory synaptic connections to the VGAT-positive neurons within the pre-BötC .', 'However , our results indicate that these possible inhibitory effects were small , and therefore the optogenetic experimental results obtained in our study in slices in vitro were always attributed to excitatory effects ( i . e . , membrane depolarization ) on VGAT-positive inspiratory neurons within the pre-BötC ( as in Fig 3C ) .', 'To investigate the role of inhibitory neurons within the pre-BötC in the generation and control of the respiratory pattern , we used site-specific optogenetic stimulations of VGAT expressing inhibitory neurons by single laser pulses of different durations ( 0 . 3–1 . 0 s ) and power ( 1 . 0 or 2 . 0 mW ) delivered bilaterally to both ( left and right ) pre-BötC regions at different phases of the respiratory cycle .', 'The perturbations of respiratory neural activity induced by these photostimulations were dependent on the phase of application and the intensity of the applied laser pulses .', 'Low intensity ( 1 . 0 mW ) single pulses of 300 ms duration delivered to the pre-BötC during the inspiratory phase did not affect the respiratory rhythm ( n = 9 , Fig 4A and 4G ) .', 'In contrast , the same low-intensity stimuli outlasting the inspiratory phase ( n = 6 , Fig 4B ) or applied during the expiratory phase ( n = 6 , Fig 4C ) , induced rebound activation and thus an advanced onset of the next inspiratory phase .', 'Only when the stimulus occurred very late during the expiratory phase and lasted into the next inspiratory phase , inspiration was delayed until the light stimulus was turned off ( n = 6 , Fig 4D ) .', 'The dependence of the stimulation effect on the respiratory phase was abolished when stronger stimuli ( 2 . 0 mW ) where applied .', 'The 2 . 0 mW laser pulses applied to the pre-BötC during the inspiratory phase reliably terminated the current inspiratory burst ( n = 9 , Fig 4G ) and initiated rebound activation of inspiration leading to an advanced onset of the next inspiratory burst ( n = 9 , Fig 4E ) .', 'Similarly , such stronger stimuli delivered during the expiratory phase caused rebound activation and advanced onset of the next inspiratory burst ( n = 6 , Fig 4F ) .', 'Rebound activation , whenever it occurred , showed a consistent latency of 300 ms on average that was independent of the stimulation intensity ( n = 6 , Fig 4H ) .', 'To investigate how a sustained stimulation of pre-BötC inhibitory neurons affects the respiratory activity , we applied epochs ( duration 10–60 s ) of sustained laser illumination ( 20 Hz , 20 ms pulse trains , laser power of 0 . 5–5 . 0 mW ) allowing a recovery period ( > 4min ) after each epoch .', 'Bilateral laser illumination of pre-BötC regions caused rapid and reversible perturbations of the respiratory frequency ( monitored by phrenic nerve ( PN ) or pre-BötC inspiratory activity recordings ) as a function of laser power ( 0 . 5–5 . 0 mW range ) .', 'Relatively weak sustained stimulation ( 0 . 5–2 . 0 mW ) increased respiratory frequency ( Fig 5A for a representative example of 2 . 0 mW stimulation ) .', 'With further increase in the stimulation intensity , the frequency started to decrease ( Fig 5D ) .', 'The respiratory frequency was significantly reduced at 4 . 0 mW ( Fig 5B ) and the respiratory rhythm was abolished at a stimulation intensity of 5 . 0 mW ( Fig 5C ) .', 'The relationship between the respiratory frequency and stimulation intensity is illustrated in Fig 5D ( n = 10 , mean ± SEM , **p < 0 . 01 , *p < 0 . 05 ) showing that bilateral activation of inhibitory neurons in the pre-BötC region affects the respiratory frequency in a photostimulation intensity-dependent manner .', 'At relatively low stimulation intensity ( laser power of 0 . 5–2 . 0 mW ) frequency increased monotonically ( linear regression fit: slope = 27 . 63 ± 7 . 39% per mW , r = 0 . 80 ) , However , no significant increase of respiratory frequency was observed at 3 . 0 mW , and the stronger laser stimulation ( 4 . 0–5 . 0 mW ) caused a significant decrease of frequency with termination of respiratory activity for the duration of the photostimulation at the maximum laser power ( 5 . 0 mW ) .', 'To investigate the involvement of the BötC inhibitory neurons in the generation and control of the respiratory pattern , we applied bilateral site-specific photostimulation of VGAT expressing BötC neurons .', 'In contrast to the intensity- and phase-dependent effects of pre-BötC stimulations , the effect of single light pulses applied to the BötC terminated inspiratory activity with photostimulation at low laser power ( 1 . 0 mW , n = 5 , Fig 6 ) , independent of the phase of application .', 'The respiratory activity was terminated for the entire duration of the photostimulation epoch ( Fig 6A–6D ) and a release of light stimulation induced rebound activation of inspiration after a fixed latency of approximately 300 ms ( Fig 6E ) , very similar to the rebound activation after stimuli applied to the pre-BötC .', 'Sustained optogenetic activation of BötC ChR2-expressing inhibitory neurons ( 20 Hz pulses , 0 . 5–3 . 0 mW , 30–60 sec duration ) caused a rapid and reversible reduction of inspiratory frequency ( Fig 7A for a representative example of the effect of 2 . 0 mW stimulation ) with complete cessation of the rhythm at a maximum laser power of 3 . 0 mW ( Fig 7B ) .', 'Fig 7C ( n = 12 , mean ± SEM , ** p < 0 . 01 ) illustrates that bilateral sustained activation of BötC inhibitory neurons monotonically reduced inspiratory frequency in a laser power-dependent manner ( 0 . 5–3 . 0 mW , linear regression fit: slope = -35 . 36 ± 2 . 28% per mW , r = -0 . 89 ) .', 'To investigate the effects of photostimulation ( 20 Hz pulses , 0 . 5–3 . 0 mW ) in the BötC region on the activity of BötC and pre-BötC respiratory neurons , we recorded extracellular population activity from BötC post-I or aug-E type of expiratory neurons as well as pre-BötC pre-I/I type inspiratory neurons in perfused brainstem-spinal cord in situ preparations from adult VGAT-ChR2 mouse lines .', 'Extracellular recordings from post-I expiratory neurons in the BötC ( n = 3 , Fig 8A ) showed a rapid and reversible augmentation , typically tonic excitation of neuron activity during laser application at ≥ 2 mW laser power to the BötC , which also caused reduction of inspiratory frequency and inhibition of inspiratory PN activity .', 'In aug-E type of expiratory neurons in the BötC ( n = 4 , Fig 8B ) , photostimulation to the BötC similarly caused augmentation of neuron activity along with reduction of inspiratory frequency and inhibition of inspiratory PN activity .', 'These results indicate that laser illumination ( 0 . 5–3 . 0 mW ) in the BötC region induced excitatory effects on expiratory VGAT-positive inhibitory neurons in the BötC .', 'On the other hand , extracellular recordings from inspiratory ( pre-I/I ) neuron activity in the pre-BötC ( n = 6 , Fig 8C ) showed inhibition , typically complete silencing , of neuron activity during laser application to the BötC , which also caused a reduction of inspiratory frequency and inhibition of inspiratory PN activity .', 'These results suggest functional inhibitory connections from BötC to pre-BötC circuits , consistent with the core respiratory network configuration proposed in our computational model ( see below ) .', 'Previous models of the brainstem respiratory circuits [3 , 4 , 6–8 , 10] proposed neural interactions within and between the pre-BötC and BötC as shown in Fig 9A .', 'In these models , the pre-BötC contained only populations of inspiratory neurons: the excitatory pre-inspiratory/inspiratory ( pre-I/I ) and the inhibitory early-inspiratory ( early-I ) populations .', 'The BötC contained the inhibitory post-inspiratory ( post-I ) and augmenting expiratory ( aug-E ) populations .', 'Hence , post-I neurons were suggested to be only present in the BötC but not in the pre-BötC .', 'However , our analysis has shown that this architecture of the pre-BötC/BötC core network is unable to reproduce and explain the above experimental data on phase- and stimulation intensity-dependent effects of site-specific activation of inhibitory neurons within the pre-BötC .', 'This conclusion was based on the following .', 'First , optogenetic stimulation of the pre-BötC inhibitory neurons revealed a rebound activation of inspiration after the end of a sufficiently strong photostimulus ( Fig 4B–4F ) .', 'The excitatory pre-I/I population could , in principle , generate a rebound activation , because of the persistent sodium-dependent mechanism .', 'However , the pre-BötC compartment in previous models contained only the early-I inhibitory population ( Fig 9A ) which was active during inspiration only , did not inhibit pre-I/I neurons , and thus could not cause rebound activation of pre-I/I neurons .', 'Second , we showed that the effect of low-intensity photostimulation of inhibitory pre-BötC neurons was suppressed during inspiration ( Fig 4A ) , but this suppression could be overcome by a stronger light activation ( Fig 4E ) .', 'Together these observations suggest that the neurons responsible for termination of inspiration could be inhibited during the inspiratory phase unless the intensity of laser stimulation overcame this inhibition .', 'Finally , additional activation of early-I inhibitory neurons in the previous models was known to increase respiratory frequency ( e . g . [10] ) and thus could not account for the biphasic shape of the frequency response to sustained light activation of inhibitory pre-BötC neurons ( Fig 5A ) .', 'Further analysis of the previous models in the context of our new data led us to the suggestion that the above effects can be reproduced and explained by having post-inspiratory neurons located in both the BötC and the pre-BötC .', 'Indeed , neurons with post-inspiratory firing characteristics have been previously found in the pre-BötC [9 , 26 , 27] .', 'Therefore , we extended the previous models by incorporating an additional population of post-inspiratory inhibitory neurons in the pre-BötC and called it post-IpBC , ( Fig 9B ) .', 'Except for its location , the proposed inhibitory post-IpBC population was similar to the post-I population in the BötC: it had adapting intrinsic properties and inhibited all other populations in the network except for its partner post-I population in the BötC .', "Like the BötC's post-I population , the post-IpBC population had mutual inhibitory interactions with the early-I population of the pre-BötC and the aug-E population of the BötC ( Fig 9B ) .", 'To match model behavior to the experimental data , we assumed that the post-IpBC strongly inhibits the pre-I/I and other respiratory populations leading to rebound excitation of inspiration after ending the post-IpBC photostimulation ( Fig 4B–4D ) .', 'The dependence of network behavior on the intensity of the applied photostimulation led us to the suggestion that the observed different ( and sometimes opposite ) consequences of weak vs . strong stimulations may result from mutual inhibitory interactions between the post-IpBC and early-I populations of the pre-BötC , so that the early-I inhibits post-IpBC at lower stimulation intensities , whereas post-IpBC overcomes this inhibition at higher stimulation intensities .', 'Activation of ChR2 by photostimulation was implemented in the model as opening an additional excitatory "ChR2" channel in the populations of inhibitory neurons within the compartment ( pre-BötC or BötC ) stimulated by light .', 'The light sensitivity of each inhibitory population was defined by the conductance of this channel ( gChR , which was the same in each inhibitory population , see Methods ) .', 'The intensity of light stimulation was characterized by the parameter “stimChR“ .', 'Incorporating the gChR , conductance in all inhibitory neurons allowed the above mutual inhibitory interactions between the post-IpBC and early-I populations .', 'Specifically , at relatively low stimulation intensity during inspiration , the early-I population ( receiving excitation from the pre-I/I population ) kept the post-IpBC population inhibited , and thus did not allowing the applied stimulation to terminate inspiration .', 'In this case , the early-I population became highly active and close to saturation of its activity .', 'Consequently , an additional increase of stimulation intensity affected early-I activity less than that of the post-IpBC population .', 'These different degrees of additional activation by photostimulation allowed the post-IpBC population to overcome early-I inhibition at higher stimulation intensity and to inhibit both early-I and pre-I/I populations , hence terminating inspiration .', 'Network operation under normal conditions is shown in Fig 9C .', 'During expiration , the BötC post-I population demonstrates adapting ( decrementing ) activity ( see Methods ) .', 'The corresponding decline in inhibition from this population shapes the augmenting profile of aug-E activity .', 'The reduction in post-I inhibition also produces slow depolarization of pre-I/I and early-I neurons in the pre-BötC .', 'In addition , the pre-I/I population is depolarizing because of the slow deinactivation of the persistent ( slowly inactivating ) sodium current ( INaP , see Methods ) .', 'At some moment during expiration , the pre-I/I population rapidly activates , providing excitation of early-I .', 'The latter inhibits post-I , aug-E and post-IpBC populations , completing the switch from expiration to inspiration .', 'During inspiration , the early-I population of the pre-BötC demonstrates adapting ( decrementing ) activity .', 'The decline in inhibition from this population produces a slow depolarization of the post-I , aug-E and post-IpBC populations .', 'At some moment , the post-I populations of BötC rapidly activates and inhibits both inspiratory neurons ( pre-I and early-I ) and the aug-E neuron ( initially ) , producing the switch from inspiration to expiration and the release of the fast adapting post-IpBC population in the pre-BötC .', 'The model reproduced our experimental findings on the effects of selective optogenetic activation of inhibitory neurons in the pre-BötC ( Fig 10 ) .', 'Similar to our experimental data , activating both inhibitory populations in the pre-BötC ( early-I and post-IpBC ) in the model by the simulated weak pulses ( 300 ms , stimChR = 1 ) did not terminate inspiration and did not affect the respiratory rhythm ( Fig 10A ) .', 'The weak pulses induced rebound activation advancing the next inspiratory activity if the stimulus outlasted the inspiratory phase ( 1 s , stimChR = 1 , Fig 10B ) or was applied during the expiratory phase ( 300 ms , stimChR = 1 , Fig 10C ) .', 'Also , inspiratory phase activity was delayed until the light stimulus was turned off , when the stimulus occurred very late during the expiratory phase and lasted into the next inspiratory phase ( 300 ms , stimChR = 1 , Fig 10D ) .', 'Similar to our experimental data , stronger stimuli ( 300 ms , stimChR = 2 ) applied during the inspiratory phase terminated inspiratory activity ( Fig 10E ) .', 'Strong stimuli during either the inspiratory or expiratory phase initiated rebound activation of inspiration and advanced the next inspiratory activity phase ( Fig 10E and 10F ) .', 'As seen in Fig 10B–10F termination of the stimulus first produced a release of the aug-E population of the BötC , whose activity created a fixed latency for release of activity of the pre-BötC pre-I/I population that defines the inspiratory phase .', 'The model also reproduced the biphasic frequency response of a sustained activation of inhibitory neurons within the pre-BötC .', 'Sustained activation of early-I and post-IpBC inhibitory populations increased the frequency at low stimulation intensities ( stimChR ≤ 1 , Fig 11A ) , whereas further increase of stimulus intensity reduced the frequency ( stimChR > 1 , Fig 11B ) and finally terminated respiratory oscillations ( stimChR > 1 . 8 , Fig 11C ) .', 'The intensity range of the sustained stimulation in the model was reduced by a factor of two ( Fig 11C ) , which is consistent with the level of inactivation under continuous light stimulation of ChR2 channels in the biological system [49 , 50] .', 'Similar to our experimental studies , sustained activation of inhibitory neurons within the BötC ( aug-E and post-I ) by short ( 300 ms ) and long ( 5 s ) single pulses inhibited the respiratory rhythm for the duration of each pulse ( Fig 12 ) and the termination of each stimulus first produced a transient release of activity of the BötC aug-E population which delayed the onset of the next inspiratory phase with a latency ( Fig 12A–12D ) consistent with our experimental studies .', 'Also , similar to the experimental results , a sustained activation of inhibitory populations within the BötC decreased the respiratory frequency with low stimulation intensities ( stimChR < 0 . 18 , Fig 13A and 13C ) and suppressed all activity with higher intensities ( stimChR ≥ 0 . 18 , Fig 13B and 13C ) , albeit at a lower intensity range when compared to the short pulses and to the pre-BötC stimulation .', 'The computational model presented here reproduced our experimental findings on the effects of sustained and phase-dependent short-duration activation of inhibitory neurons in the pre-BötC .', 'To validate the model performance further , we simulated the simultaneous optogenetic stimulation of all , inhibitory and excitatory , neurons within the pre-BötC and compared the modeling results with findings in a recently study of Alsahafi et al . [47] .', 'They unilaterally activated pre-BötC or BötC neurons using an adeno-associated virus expressing ChR2 under the synapsin promoter to selectively photoactivate all ( excitatory and inhibitory ) neurons in the pre-BötC or the BötC [47] .', 'Specifically , Alsahafi et al . [47] showed that the sustained stimulation of pre-BötC increased respiratory frequency ( Fig 14A , top diagram ) and that the network could be entrained by repetitive application of short light pulses ( Fig 14B , top diagram ) .', 'To test the model against the experimental data published by Alsahafi et al . [47] we set the light sensitivity of the excitatory pre-I/I population equal to that of post-IpBC and early-I populations ( see Methods ) .', 'Similar to the experimental results of Alsahafi et al . [47] , sustained stimulation of all populations in the pre-BötC increased respiratory frequency in our model at stimChR = 0 . 18 ( Fig 14A , bottom diagram ) .', 'In the model , entrainment of the rhythm was possible down to a respiratory cycle period of 1 . 5 s with 300 ms pulses of stimChR = 0 . 2 to all populations of the model pre-BötC ( Fig 14B , bottom diagram ) .', 'Alsahafi et al . [47] also demonstrated that optogenetic stimulation of pre-BötC neurons with short stimuli could induce a reliable reset of inspiratory activity but found a post-inspiratory period during which the rhythm could not be reset ( Fig 15A ) .', 'The reset of inspiration in their experiments occurred with a short delay ( about 100 ms ) after the stimulus onset .', 'The post-inspiratory period during which optogenetic stimulation of the pre-BötC was unable to initiate the next inspiratory event was about 200 ms ( Fig 15A ) .', 'In our model , stimulating all neuron populations within the pre-BötC also initiated a reset of the inspiratory burst ( with a slightly longer latency of ~200 ms ) when stimulated at stimChR = 0 . 2 ( Fig 15B ) .', 'The post-I period insensitive for inspiratory resetting was about 300 ms and was mainly defined by the activity of the post-I population of the BötC and the time constant of its adaptation .', 'Our experimental and modeling results were also consistent with other data reported by Alsahafi et al . [47] .', 'In some experiments , they used optogenetic excitation of BötC neurons which resulted in a decrease in respiratory frequency or complete suppression of inspiratory activity during the applied stimuli ( Fig 4B and 4C in their paper ) .', 'This corresponds to our experimental ( Fig 7 ) and modeling ( Fig 11 ) results .', 'Thus , our model reproduces multiple results of experimental perturbations that employed selective optogenetic activation of either inhibitory neurons within the pre-BötC and BötC or of all neuron types in each of these compartments .'], ['We established a VGAT-tdTomato-ChR2-EYFP transgenic mouse line with Cre-driven ChR2 expression selectively in VGAT-positive neurons for temporally con
['Introduction', 'Results', 'Discussion', 'Methods']
['The circuit organization within the mammalian brainstem respiratory network , specifically within and between the pre-Bötzinger ( pre-BötC ) and Bötzinger ( BötC ) complexes , and the roles of these circuits in respiratory pattern generation are continuously debated .', 'We address these issues with a combination of optogenetic experiments and modeling studies .', 'We used transgenic mice expressing channelrhodopsin-2 under the VGAT-promoter to investigate perturbations of respiratory circuit activity by site-specific photostimulation of inhibitory neurons within the pre-BötC or BötC .', 'The stimulation effects were dependent on the intensity and phase of the photostimulation .', 'Specifically: ( 1 ) Low intensity ( ≤ 1 . 0 mW ) pulses delivered to the pre-BötC during inspiration did not terminate activity , whereas stronger stimulations ( ≥ 2 . 0 mW ) terminated inspiration .', '( 2 ) When the pre-BötC stimulation ended in or was applied during expiration , rebound activation of inspiration occurred after a fixed latency .', '( 3 ) Relatively weak sustained stimulation ( 20 Hz , 0 . 5–2 . 0 mW ) of pre-BötC inhibitory neurons increased respiratory frequency , while a further increase of stimulus intensity ( > 3 . 0 mW ) reduced frequency and finally ( ≥ 5 . 0 mW ) terminated respiratory oscillations .', '( 4 ) Single pulses ( 0 . 2–5 . 0 s ) applied to the BötC inhibited rhythmic activity for the duration of the stimulation .', '( 5 ) Sustained stimulation ( 20 Hz , 0 . 5–3 . 0 mW ) of the BötC reduced respiratory frequency and finally led to apnea .', 'We have revised our computational model of pre-BötC and BötC microcircuits by incorporating an additional population of post-inspiratory inhibitory neurons in the pre-BötC that interacts with other neurons in the network .', 'This model was able to reproduce the above experimental findings as well as previously published results of optogenetic activation of pre-BötC or BötC neurons obtained by other laboratories .', 'The proposed organization of pre-BötC and BötC circuits leads to testable predictions about their specific roles in respiratory pattern generation and provides important insights into key circuit interactions operating within brainstem respiratory networks .']
['We investigated the organization of key circuits within the brainstem respiratory central pattern generator ( CPG ) responsible for generating the basic breathing pattern in mammals .', 'Our study focused on the organization and interactions of two core components of this CPG: the pre-Bötzinger ( pre-BötC ) and Bötzinger ( BötC ) complexes .', 'We used genetically transformed mice with inhibitory neurons containing light activated membrane channels , allowing us to investigate perturbations of respiratory activity during site-specific activation of pre-BötC or BötC inhibitory neurons by applying laser photostimuli of different intensities and durations at different phases of the respiratory cycle .', 'Photostimulation of inhibitory neurons caused major disturbances of circuit activity .', 'The obtained experimental results were used to extend and refine a computational model simulating circuit interactions within and between the pre-BötC and BötC .', 'A major feature of the model is a novel inhibitory post-inspiratory population within the pre-BötC and its interactions with other populations in the network .', 'We validated the model by successfully testing its ability to reproduce our experimental results as well as results of previous studies that employed photoactivation of pre-BötC or BötC neurons .', 'Our study provides important insights into the circuit organization in the brainstem respiratory CPG with implications for other circuits controlling rhythmic behaviors .']
['medicine and health sciences', 'neurochemistry', 'optogenetics', 'chemical compounds', 'neural networks', 'engineering and technology', 'aliphatic amino acids', 'lasers', 'brain', 'light', 'neuroscience', 'organic compounds', 'electromagnetic radiation', 'brain mapping', 'amino acids', 'bioassays and physiological analysis', 'optical equipment', 'neurotransmitters', 'research and analysis methods', 'glycine', 'computer and information sciences', 'animal cells', 'proteins', 'chemistry', 'light pulses', 'brainstem', 'neurophysiological analysis', 'gamma-aminobutyric acid', 'physics', 'biochemistry', 'cellular neuroscience', 'cell biology', 'anatomy', 'organic chemistry', 'equipment', 'neurons', 'biology and life sciences', 'cellular types', 'physical sciences']