perturbations of respiratory rhythm and pattern by ... · ratory rhythm and motor pattern...

Sensory and Motor Systems

Perturbations of Respiratory Rhythm and Patternby Disrupting Synaptic Inhibition withinPre-Bötzinger and Bötzinger Complexes1,2,3

Vitaliy Marchenko,1,� Hidehiko Koizumi,2,� Bryan Mosher,2 Naohiro Koshiya,2 Mohammad F. Tariq,2

Tatiana G. Bezdudnaya,1 Ruli Zhang,2 Yaroslav I. Molkov,3 Ilya A. Rybak,1 and Jeffrey C. Smith2

DOI:http://dx.doi.org/10.1523/ENEURO.0011-16.2016

1Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, Pennsylvania 19129,2Cellular and Systems Neurobiology Section, National Institute of Neurological Disorders and Stroke, NationalInstitutes of Health, Bethesda, Maryland 20892, and 3Department of Mathematics and Statistics, Georgia StateUniversity, Atlanta, Georgia 30302

Visual Abstract

The pre-Bötzinger (pre-BötC) and Bötzinger (BötC) complexes are the brainstem compartments containinginterneurons considered to be critically involved in generating respiratory rhythm and motor pattern in mammals.Current models postulate that both generation of the rhythm and coordination of the inspiratory-expiratory patterninvolve inhibitory synaptic interactions within and between these regions. Both regions contain glycinergic and

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GABAergic neurons, and rhythmically active neurons in these regions receive appropriately coordinated phasicinhibition necessary for generation of the normal three-phase respiratory pattern. However, recent experimentsattempting to disrupt glycinergic and GABAergic postsynaptic inhibition in the pre-BötC and BötC in adult rats invivo have questioned the critical role of synaptic inhibition in these regions, as well as the importance of the BötC,which contradicts previous physiological and pharmacological studies. To further evaluate the roles of synapticinhibition and the BötC, we bilaterally microinjected the GABAA receptor antagonist gabazine and glycinergicreceptor antagonist strychnine into the pre-BötC or BötC in anesthetized adult rats in vivo and in perfused in situbrainstem–spinal cord preparations from juvenile rats. Muscimol was microinjected to suppress neuronal activityin the pre-BötC or BötC. In both preparations, disrupting inhibition within pre-BötC or BötC caused majorsite-specific perturbations of the rhythm and disrupted the three-phase motor pattern, in some experimentsterminating rhythmic motor output. Suppressing BötC activity also potently disturbed the rhythm and motorpattern. We conclude that inhibitory circuit interactions within and between the pre-BötC and BötC criticallyregulate rhythmogenesis and are required for normal respiratory motor pattern generation.

Key words: Bötzinger complex; brainstem; central pattern generation; pre-Bötzinger complex; respiration;synaptic inhibition

IntroductionRhythmic movements such as breathing and locomo-

tion are produced by central pattern generator (CPG)networks containing interacting excitatory and inhibitorycircuits. These circuits are the neural substrates for pro-ducing motor behavior (Grillner, 2006) and defining theirspecific roles in rhythmic motor pattern generation is keyto understanding the functional operation of CPGs. Herewe have addressed the longstanding problem of definingroles of inhibitory circuits in core structures of the mam-malian brainstem respiratory CPG.

The respiratory neural pattern under normal conditionsincludes three phases: inspiration, post-inspiration, andlate expiration (Richter, 1996; Richter and Smith, 2014).The kernel of the respiratory CPG located in the ventralrespiratory column of the medulla includes two key com-partments, the pre-Bötzinger (pre-BötC) and Bötzinger(BötC) complexes (Alheid and McCrimmon, 2008; Smithet al., 2009, 2013). The pre-BötC contains a heteroge-neous population of excitatory neurons, including cellswith intrinsic bursting properties, and excitatory synapticinterconnections that generate rhythmic inspiratory activ-ity and drive inspiratory motor output. This excitatorypopulation, when isolated in slices in vitro, generatesinspiratory bursting activity (Smith et al., 1991; Koshiyaand Smith, 1999) that persists after disrupting synapticinhibition (Johnson et al., 2001). In addition, the pre-BötCcontains GABAergic and glycinergic neuron populations(Kuwana et al., 2006; Winter et al., 2009; Morgado-Valleet al., 2010; Koizumi et al., 2013) providing phasic inspira-tory inhibition widely distributed in the brainstem includinginhibition of BötC expiratory neurons. In turn, BötC post-inspiratory and expiratory neurons provide phasic expira-tory inhibition (Jiang and Lipski, 1990; Tian et al., 1999a,b;Ezure et al., 2003a,b) including to pre-BötC inspiratoryneurons to coordinate generation of expiratory and in-spiratory phases.

The specific contributions of the intrinsic excitatorybursting mechanisms in the pre-BötC, and inhibitory net-

Received January 16, 2016; accepted April 18, 2016; First published May 02,2016.1The authors report no conflict of interest.2Author contributions: V.M., H.K., I.A.R., and J.C.S. designed research;

V.M., H.K., B.M., T.G.B., and R.Z. performed research; H.K., N.K., M.F.T., R.Z.,and Y.I.M. analyzed data; I.A.R. and J.C.S. wrote the paper.

3This work was supported in part by the Intramural Research Program of theNational Institutes of Health (NIH), National Institute of Neurological Disordersand Stroke, and NIH Grants R01 NS069220 and R01 AT008632

*V.M. and H.K. contributed equally to this work.Correspondence should be addressed to Dr. Jeffrey C. Smith, 49 Convent

Drive, Room 2A10, NINDS, NIH, Bethesda, MD 20892. E-mail: [email protected].

DOI:http://dx.doi.org/10.1523/ENEURO.0011-16.2016Copyright © 2016 Marchenko et al.This is an open-access article distributed under the terms of the CreativeCommons Attribution 4.0 International, which permits unrestricted use, distri-bution and reproduction in any medium provided that the original work isproperly attributed.

Significance Statement

Defining functional roles of postsynaptic inhibition in respiratory and other mammalian central patterngeneration circuits is a longstanding problem. Inhibitory circuit interactions within and between thebrainstem respiratory pre-BötC and BötC have been proposed to be critically involved in normal rhythm andmotor pattern generation. A fundamental role of postsynaptic inhibition in these regions has been ques-tioned in recent experiments attempting to pharmacologically disrupt this inhibition. To resolve thiscontradiction, we applied similar approaches of microinjecting selective pharmacological antagonists ofGABAAergic and glycinergic receptor-mediated inhibition in the pre-BötC and BötC in rats. Our resultsdemonstrate large, site-specific perturbations of respiratory rhythm and motor pattern including disruptionof rhythmic motor output and thus confirm the critical role of inhibitory circuit interactions.

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work interactions between pre-BötC and BötC, to respi-ratory rhythm and motor pattern generation are not clearlyunderstood and this issue is continuously debated. Earlytheoretical models, based entirely on network inhibitoryinteractions, could not explain the maintenance of rhythmafter blockade of synaptic inhibition in vitro. Alternatively,the pure autorhythmic excitatory network models, devel-oped to explain the in vitro data, could not account formany behaviors observed in vivo, such as the Hering–Breuer inspiratory inhibitory and other respiratory reflexesand the coordinated generation of multiple respiratoryphases. Also, these models could not reproduce apneu-sis, a breathing pattern characterized by a significantlyprolonged inspiration alternating with short expiratory in-tervals (Lindsey et al., 2012).

To resolve this problem, more complicated modelshave been developed (Rybak et al., 2004, 2007; Smithet al., 2007, 2009, 2013) hypothesizing that: (1) the pre-BötC, although capable of autonomous generation ofrhythmic bursting when isolated in vitro, is embeddedin the larger respiratory network where its activity iscontrolled by interactions with other brainstem com-partments, including inputs from excitatory RTN/pFRGneurons, and from the inhibitory neuron populations inBötC, and (2) both the intrinsic bursting of pre-BötC in-spiratory neurons and inhibitory interactions between theneural populations in pre-BötC and BötC are fundamen-tally involved in generating the normal rhythmic respira-tory pattern. Disruption of inhibition in these circuits wouldlead either to switching to the intrinsic rhythmic activityoriginating within the pre-BötC, or to sustained orapneustic-like activity.

This concept was challenged by a recent study in theanesthetized rat using targeted pharmacological block-ade of fast inhibitory neurotransmission (Janczewskiet al., 2013) from which it was concluded that: (1) the BötCdoes not play a role in respiratory rhythm/pattern gener-ation, and (2) inhibition within the pre-BötC and BötC isnot required for generating a normal breathing rhythm andpattern.

The present study was focused on resolving this con-tradiction and further evaluating roles of inhibitory inter-actions in pre-BötC and BötC in rhythm generation andshaping respiratory pattern. Our experiments were per-formed using two distinct preparations, the anesthetized,vagotomized adult rat, and the arterially perfused in situbrainstem–spinal cord preparation of juvenile rat. Specificpharmacological blockers of glycinergic (strychnine) andGABAAergic (gabazine) receptor-mediated inhibition wereselectively microinjected into the pre-BötC or BötC andperturbations of the respiratory frequency and phaseswere evaluated. In addition, microinjections of the GABAA

receptor agonist muscimol were used to inhibit neuralactivity in each compartment. Our results were not con-sistent with those reported by Janczewski et al. (2013).Disrupting inhibition within pre-BötC or BötC, as well asinhibiting BötC activity, caused major perturbations of therespiratory frequency and three-phase pattern. Moreover,blocking inhibition within the BötC lead to apnea confirm-ing the critical role of inhibitory interactions between pre-

BötC and BötC. Our results are consistent with previousproposals of the important role of inhibitory interactionswithin and between pre-BötC and BötC in generating andshaping the respiratory pattern.

Materials and MethodsAnimal procedures

All experimental procedures used in this study wereapproved by either the NINDS Animal Care and UseCommittee, or the Drexel University Institutional AnimalCare and Use Committee, which oversees Drexel Univer-sity’s AAALAC International-accredited animal program.All electrophysiological recording and pharmacologicalmicroinjection experiments were performed via a sur-gically exposed ventral brainstem for access to theventrolateral medullary pre-BötC and BötC regions(Fig. 1).

Surgical procedures in adult rats in vivoSpontaneously breathing, adult male Sprague-Dawleyrats (340 –380 g) were anesthetized with isoflurane va-porized in O2 (Matrix; 4 –5% induction, 1.75–2.0%maintenance) via a snout mask. Anesthetic depth wasmaintained at a level at which withdrawal reflexes, as wellas changes in heart rate and blood pressure in responseto pinching the distal hind limbs, were absent. After tra-cheotomy with a glass tube, animals were artificially ven-tilated with the same gas mixture (60 min�1, 2.5–3.0 mltidal volume; Columbus Apparatus rodent ventilator).Electrocardiogram was measured via three small subcu-taneous electrodes using conventional amplification andfiltering (Neurolog; Digitimer) and monitored using an au-dio amplifier (model AM10; Grass Instruments) and oscil-loscope (Tektronix). One femoral artery and vein werecannulated for measurement of arterial pressure and in-fusion of drugs/saline, respectively. During all surgicalprocedures, rectal temperature was maintained at 37.0 �0.1°C via a servo-controlled heating blanket coupled to arectal thermometer (Harvard Apparatus). The phrenicnerve (PN) was prepared for recording by dissecting thenerves free from the surrounding tissue. Ventral neckmuscles (cleidomastoideus, sternomastoideus, sternohy-oideus, omohypides, and digastricus), infrathyroid por-tions of the trachea and esophagus were removed. Thebody of the 1st neck vertebra (atlas) and base portion ofoccipital bone were removed to expose the ventralmedulla and the axo-occipital membrane was cut. Thedura was then opened using iridectomy scissors andresidual bleeding from lateral epidural sinus was ar-rested by applying small pieces of gelfoam (USP, Phar-macia) soaked with thrombin solution (50 U · ml�1 USP,Biopharm Laboratories) dissolved in artificial CSF(aCSF).

All animals were vagotomized and baro- and chemore-ceptor denervated via bilateral transection of the carotidsinus nerves to prevent cardiorespiratory reflex influenceson motor nerve outputs (Richter and Seller, 1975; Grundyet al., 1986; Hopp and Seagard, 1998; Virkki et al., 2007;Baekey et al., 2010). A bilateral pneumothorax was per-formed before electrophysiological recording to eliminate



lung inflation-related movement artifacts and chest wallmechanoreceptor feedback. A positive end-expiratorypressure of 1.0 cm H2O was maintained to prevent lungatelectasis during expiration. Animals were paralyzed by

an intravenous bolus injection (2 mg/kg), followed bycontinuous infusion (3–4 mg/kg/h), of vecuronium bro-mide (Abbott Laboratories) dissolved in Ringer–Locke so-lution. End-tidal CO2 was maintained between 5.0% and

Figure 1. Ventral view of the adult rat medulla and histology illustrating targeted sites for pharmacology experiments in vivo. A,Photograph of adult rat brainstem ventral surface as exposed in the in vivo experimental preparations with an overview of targetedlocations (caudal to facial motor nucleus, VII) of BötC and pre-BötC as routinely identified by electrophysiological mapping of neuronalactivity profiles in the present experiments. B, Photomicrograph of ventral medullary surface and bilaterally arranged pipettes (bluedye-filled for visualization) as typically configured for near perpendicular penetrations of the ventral surface for simultaneousmicroinjections. C, D, Confocal microscopic images of parasagittal histologic sections (50 �m thick) showing, respectively, examplesof targeted sites for microinjection of inhibitory antagonists in the pre-BötC ventral to the semicompact subdivision of nucleusambiguous (NAsc), and in the BötC ventral to the compact subdivision of nucleus ambiguus (NAc). Targeted sites are marked bymicroinjected solution of fluorescent microspheres (green). NA and VII motoneurons are immunolabeled by ChAT antibody (red).Rostrocaudal spatial extent of the pre-BötC and BotC compartments are indicated. vs, ventral surface.



5.5% (Capstar, CWE) by adjusting frequency of ventila-tion. If necessary, animals were continuously infused withRinger–Locke solution (1.0–1.25% body weight or 10.0–12.5 ml/kg/h) to maintain a stable mean arterial pressureof 85–95 mmHg.

In situ arterially perfused brainstem–spinal cordpreparationExperiments were also performed with the in situ arteriallyperfused brainstem–spinal cord preparations from juve-nile rats (3–5 weeks old) as previously described (Paton,1996; Smith et al., 2007). These preparations were stud-ied because they provide the opportunity to investigateroles of synaptic inhibition in an un-anesthetized prepa-ration generating the three-phase respiratory pattern thatcould be clearly identified from simultaneous recordingsof spinal and cranial nerves, including prominent post-inspiratory (post-I) discharge recorded from the centralvagus nerve (cVN). We note that cVN recordings from thenormocapnic, vagotomized, carotid body denervated,and isoflurane anesthetized adult rats in vivo in our exper-iments do not routinely exhibit post-I discharge althoughneuronal recording in the BötC always shows post-Iactivity (see Fig. 3B), which is an important feature ofmedullary respiratory circuit activity. Furthermore, anumber of theoretical models (Rybak et al., 2007; Smithet al., 2007; Rubin et al., 2009; Shevtsova et al., 2011,2014; Richter and Smith, 2014) postulating roles ofpre-BötC and BötC inhibitory circuits in respiratoryrhythm and pattern generation have been based in parton experimental results obtained from these in situpreparations. It is therefore critical to test their roles inthis preparation, and to compare results from the in vivoanesthetized adult rat preparations using a similar strat-egy for targeted disruption of synaptic inhibition in thepre-BötC and BötC.

Preheparinized (1000 units, given intraperitoneally) ju-venile rats (Sprague-Dawley, 45–90 g; male) were anaes-thetized deeply with 5% isoflurane and the portion of thebody caudal to the diaphragm was removed. The headand thorax were immersed in ice-chilled carbogenatedaCSF solution (in mM: 1.25 MgSO4, 1.25 KH2PO4, 5.0 KCl,25 NaHCO3, 125 NaCl, 2.5 CaCl2, 10 dextrose, 0.1785polyethylene glycol) and the rat was decerebrated at aprecollicular level. The descending aorta, PN, and cVNwere surgically isolated. As with the in vivo preparations,ventral neck muscles, infrathyroid portions of the trachea,the esophagus, and then the 1st neck vertebra and baseportion of the occipital bone were removed to expose theventral medulla. The axo-occipital membrane was cut,and the dura was then cut open. The preparation wastransferred to a recording chamber and secured in astereotaxic head frame ventral side up. The descendingaorta was cannulated with a double-lumen catheter forperfusion and recording of perfusion pressure with a pres-sure transducer (Micron Instruments). Vecuronium bro-mide was added to the perfusate to block neuromusculartransmission (4 �g/ml; SUN Pharmaceutical Industries).The perfusate was gassed with 95% O2/5% CO2 andmaintained at 31°C. Vasopressin (200–400 pM as re-

quired; APP Pharmaceuticals) was added to the perfusateto raise and maintain perfusion pressure between 70 and80 mmHg (Paton et al., 2006). Unless stated, all chemicalswere from Sigma-Aldrich.

Electrophysiological recording in vivoWith the rat in the supine position, the central ends of thecut PN were placed on bipolar silver hook electrodes forrecording (10–5000 Hz bandpass; Neurolog, Digitimer)and immersed in a mineral oil pool formed by skin flaps.To identify precise locations of the BötC and pre-BötC theactivity of medullary expiratory and (pre)inspiratory neu-rons was recorded (200–3000 Hz bandpass; Neurolog) bya ventral approach and glass (WPI) microelectrodes (tipouter diameter of 2–3 �m, 5–10 M�) filled with 0.5 M NaCland 2% pontamine sky blue. The pre-BötC is readilyidentified by a characteristic pattern of pre-inspiratory/inspiratory (pre-I/I) activity (see Fig. 3A) and the BötC hasa characteristic profile of post-I and augmenting expira-tory (aug-E) activities (see Fig. 3B). The microelectrodewas held in a three-dimensional stepper motor assembly(DC-3K, Märzhäuser), attached to the rail of the stereo-taxic frame, and advanced in steps of 2.5–5.0 �m. Aftermapping neuronal activity, the medullary surface wasmarked bilaterally by iontophoresis for 15 min of thepontamine sky blue dye (�25 nA, 0.125 Hz, 4 s; Axoclamp2A) as a guide spot for insertion of drug microinjectionpipettes. All electrophysiological signals were recordedsimultaneously with expiratory CO2 level, arterial bloodpressure, and lung inflation pressure, on the hard disk ofa personal computer via a 16-bit analog-to-digital con-verter (PowerLab, AD Instruments, 10 kHz sampling rate)and displayed continuously with software (Chart, AD In-struments).

Electrophysiological recording in situTo monitor respiratory network activity and motor outputin the in situ perfused brainstem–spinal cord preparations,we recorded with fire-polished glass suction electrodesinspiratory activity from PN, and cVN inspiratory andpost-inspiratory activity. Signals were amplified (50,000 –100,000�; CyberAmp 380, Molecular Devices), band-pass filtered (0.3–2 kHz), digitized (10 kHz samplingrate) with an AD converter [Cambridge Electronics De-sign (CED)], and then rectified and integrated digitallywith Spike 2 software (CED). Extracellular populationactivity from pre-BötC or BötC respiratory neurons inthe perfused in situ preparations was also recordedwith a fine glass electrode (3–5 M�) filled with 0.5 M

Na� acetate.

Targeting the pre-BötC and BötC regions withmicroinjections in vivoTo block fast inhibitory transmission a cocktail of GABAA

(gabazine, Sigma-Aldrich) and glycine (strychnine, Sigma-Aldrich) receptor antagonists (in aCSF) was pressure in-jected (250 �M, 105–115 nl) into the BötC or pre-BötCbilaterally and simultaneously (1–1.5 nl/s) with small diam-eter (OD � 15 �m, ID � 7.5 �m) polished micropipettes(Drummond Scientific). In another set of experiments, theGABAA agonist muscimol was injected (100 �M, 25–30 nl)



into the BötC or pre-BötC bilaterally and simultaneously.Control injections were made at the same sites with aCSFalone. All injection volumes were measured by micro-scopically observing the change in level of the meniscuswithin the micropipette. Microinjections were done afterelectrophysiological recording to map the extracellularsingle unit/neuronal population activities characteristic ofthe pre-BötC or BötC regions as described above, andalso in some in vivo experiments after pharmacologicalprobing by microinjection of 5 nl of 10 mM L-glutamate(L-Glu) to further confirm locations of the pre-BötC orBötC regions, which have site-specific responses to brief(500 ms) local L-Glu microinjections (i.e., transient apneawith increasing arterial blood pressure (ABP) for the BötCregion, and tachypnea with decreasing ABP for the pre-BötC region; see Fig. 3C,D). These blood pressure re-sponses are typical because the pre-BötC partiallyoverlaps with the depressor caudal ventrolateral medullaand the BötC with the pressor rostral ventrolateral me-dulla (RVLM; Kanjhan et al., 1995; Lipski et al., 1996;Moraes et al., 2012). According to our mapping by extra-cellular recordings, and consistent with characteristic per-turbations obtained by our targeted L-Glu microinjectionsbased on the neuronal activity maps, BötC and pre-BötCin isoflurane anesthetized adult male rats (340–380 g)

occupy restricted areas: 1.8–2.1 mm lateral, 550–850 �mdepth from the ventral surface, and extending �400 �m inthe rostrocaudal dimension (i.e., �800–1200 �m from thecaudal pole of facial nucleus or �1.6–2.0 mm rostral toobex) for pre-BötC, and for BötC 1.9–2.2 mm lateral,450–750 �m depth, and 600–700 �m in the rostrocaudaldimension (�100–750 �m from the caudal pole of facialnucleus or �2.0–2.75 mm rostral to obex). At the end ofeach experiment fluorescent marker yellow-green mi-crobeads (0.1 �m diameter, 2% solution, Invitrogen) wereinjected via a pipette (OD � 30 �m, ID � 15 �m; Drum-mond) into the site of microinjections for post hoc mor-phological verification (Figs. 1, 2).

Because the locations of pre-BötC and BötC in adultrats relative to the ventral surface are shallow (550–850and 450–750 �m, respectively), we performed tests forback leakage of injected solution along the microinjectionpipette track. Pipettes with different outer diameters(12.5–50 �m) were filled with 2% of pontamine sky blueand only large diameter pipettes (30 �m) showed sub-stantial leak of dye back to the ventral surface duringmicroinjections. Accordingly, for drug microinjections invivo we only used pipettes with 15 �m OD that neverexhibited back leak.

Figure 2. Confocal microscopic images of histologic sections illustrating post hoc validation of microinjection sites marked byfluorescent microspheres (green) in the pre-BötC or BötC regions in fixed sections from anesthetized adult rat in vivo preparations andjuvenile rat in situ brainstem–spinal cord preparations. A, B, Coronal sections (30 �m thick) of fixed tissue at the level of pre-BötC (A)and BötC (B) from in vivo preparations. C, D, Coronal sections (30 �m) of fixed tissue at pre-BötC (C) and BötC (D) levels from juvenilerat in situ brainstem–spinal cord preparations. Subdivisions of nucleus ambiguus (NAc, compact subdivision; NAsc, semi-compactsubdivision), labeled with ChAT antibody (red), provide regional landmarks for pre-BötC (ventral to NAsc) and BötC (ventral to NAc)levels of the medulla. Each image is taken from serial coronal histologic sections obtained from experiments targeting these regions.Labeling with microinjected solution of fluorescent microbeads, with varying extent of local spread, indicates the approximate centerof the drug microinjection sites in these examples. vs, ventral surface.



Microinjections in arterially perfusedbrainstem–spinal cord preparations in situAs in vivo, we bilaterally microinjected a cocktail of gaba-zine and strychnine and in some experiments muscimol(all drugs dissolved in the perfusate aCSF) with micropi-pettes (ID � 20 �m) positioned in the pre-BötC or BötCthrough the ventral medullary surface with microdrives(Marzhauser) after mapping locations of these regionswith extracellular recording of pre-inspiratory (pre-BötC)or expiratory (BötC) neuronal population activities. Slow,continuous microinjection was accomplished by applyinglow pressure (30 mm Hg) to the pipettes with a pressurecontrol and measurement system. In preliminary experi-ments we determined that, compared with the in vivoexperiments, lower concentrations of drugs (30 �M ofgabazine and strychnine or 10 �M muscimol) bilaterallyinjected into either of these regions caused rapid andlarge disturbances of respiratory motor output and henceall experiments with the perfused brainstem–spinal cordpreparations were conducted at these lower drug con-centrations. In some experiments, extracellular recordingsof neuronal population activity in pre-BötC (during injec-tions in BötC) or BötC (during injections in pre-BötC)regions were made during drug microinjections. Fluores-cent microbeads were microinjected to mark the sites ofdrug microinjection (Figs. 1, 2) as described above for thein vivo experiments.

Histologic verification of microinjection sitesImmediately after marking locations of drug microinjectionsites, animals in vivo were transcardially perfused with400 ml of saline (10–12°C, pH 7.4) with heparin (1000U/ml) followed by 500 ml of 4% paraformaldehyde (wt/vol)in 0.1 M PBS (10–12°C, pH 7.4). A similar procedure wasfollowed (but with smaller volumes of saline and parafor-maldehyde perfusates) for perfusion fixation of the in situbrainstem–spinal cord preparations. For both prepara-tions, the brainstem was removed, postfixed in the samefixative for 24 h at 4°C, and subsequently cryoprotectedby sequential incubation in 15% and 30% sucrose in PBSand stored overnight at 4°C in 30% sucrose, 0.1 M PBSsolution. The medulla oblongata was sectioned parasag-ittaly or coronally at 30- or 50-�m-thick sections with afreezing microtome. For fluorescence immunohistochem-istry, floating sections were incubated with 10% donkeyserum in PBS with Triton X-100 (0.3%) and subsequentlyincubated for 48–72 h at room temperature with primaryantibodies for choline acetyltransferase (ChAT; goat anti-ChAT, Millipore, 1:200) to label motoneurons. Sectionswere then rinsed with PBS and incubated for 2 h withsecondary antibodies for ChAT (donkey anti-goat-Dylight488, 1:500). Individual sections were mounted on slidesand covered with an anti-fading medium (Fluoro-Gel;Electron Microscopy Sciences). Fluorescent labeling wasvisualized with a laser-scanning confocal imaging system(Zeiss LSM 510).

Signal analyses of respiratory parametersAll automated analyses of respiratory parameters fromdigitized nerve or neuronal population activities were per-formed with IDL software (Exelis VIS). Inspiratory events

were detected from digitally rectified and integratedphrenic nerve signals via a 200 ms window moving aver-age and peak detection algorithm that calculated athreshold-based zero derivative (positive peak) point. Fol-lowing peak detection, interburst interval (IBI; inverse ofburst frequency), inspiratory time (TI) and expiratory time(TE) were measured. TI was measured as the originalintegrated burst width at 20% of the peak height abovebaseline; TE was calculated as IBI – TI. Inspiratory ampli-tude (amp) was calculated by subtracting the local base-line from the peak value. The endpoint of the parameterquantification was defined when the perturbation duringor after microinjections reached it maximum or the signalsdeclined to noise level and the program started missingpeak detections (which could appear as a quantum jumpin IBI).

The time courses of the changes in the above param-eters for a given experimental group were variable, whichwe assume resulted from variability in the times requiredfor drug diffusion to affect a sufficient number of neuronsto produce the perturbations. To represent group data,we computed the mean time courses of the parametervalues by the following procedures. For each experiment,the time courses of the parameters were extracted by a30 s window moving median up to the defined endpoint,and the parameter values for each time series were nor-malized to the computed mean values during the controlperiod (from 120 to 0 s before start of microinjection).Each time course was divided into 100 time points repre-senting 1% increments from the start of microinjection(time 0) to the endpoint. We then computed the groupmean time, and also mean values of the normalized pa-rameters, at each of these points, which were plotted andconnected by lines to represent the mean time series. Themean endpoint was plotted with its �SEM represented bycrossbars (see Figs. 4B–E, 6A–D, 7B–E, 9A–D, 13B–E,and 14D–G) and the SEM of the preceding normalizedparameter values for the group time series were repre-sented by a gray band. To determine statistical signifi-cance, the control values were compared with theendpoint values for each experiment within a group usinga two-sided Wilcoxon signed-rank test (significant p value 0.05).

In addition to quantifying the respiratory parametersindicated above, we analyzed perturbations of amplitudesand durations of individual phases of the respiratory cycle(e.g., post-I activity in cVN recordings in situ) from inte-grated, cycle phase-triggered (peri-event) neurogramsaligned at the onset of the inspiratory phase defined byPN activity. Successive cycle-triggered traces were eitheroverlaid, or represented as a colored raster plot to depicttemporal profiles of activity intensity before and during/after drug injection periods (see Figs, 10A,B, 14B).

ResultsPerturbations of respiratory rhythm and patternby targeted microinjection of l-glutamate in thepre-BötC or BötC in vivoAs described in Materials and Methods, we microin-jected L-Glu in pre-BötC and BötC regions (n � 15, 10



mM, 5 nl) to establish characteristic regional excitatoryperturbations. These in vivo experiments demonstratethat activation of pre-BötC (n � 5) by brief (500 ms)L-Glu microinjections always produced a transient in-crease of respiratory frequency (from 28.9 � 4.4 to

44.55 � 6.1 bursts/min, p 0.0001) due to a significantreduction of expiratory phase duration (TE; Fig. 3C).In contrast, the same L-Glu microinjections into BötC(n � 10) always transiently prolonged the respiratoryperiod (by 5.4 to 21 s range; mean value � 10.1 � 4.03

Figure 3. Characteristic profiles of extracellularly recorded neuronal population activity and examples of perturbations of inspiratorymotor output activity and blood pressure produced by pharmacological excitation of neurons within the pre-BötC or BötC regions inthe adult rat in vivo. A, A typical example of pre-inspiratory/inspiratory (pre-I/I) population activity used for the identification ofpre-BötC. B, An example of post-I and aug-E population activity (post-I/aug-E, simultaneously recorded in this example) used for theidentification of BötC. In both A and B, the raw recording from the phrenic nerve (PN) and PN integrated activity (�PN) are shown atthe bottom. C, A 500 ms duration microinjection of L-Glu (Glutamate) in the pre-BötC produced an increase in the �PN burstfrequency (see the trace for integrated PN activity, �PN), and a transient decrease in the ABP. The respiratory frequency (fR; bottomtrace, green) increased primarily due to reduction in expiratory phase duration (TE trace, blue) at a relatively unchanged inspiratoryduration (TI trace, red). D, A microinjection of L-Glu in the BötC caused a rapid suppression of PN activity (see traces for �PNand inspiratory, TI, and expiratory, TE, durations) accompanied by an increase of ABP. In C and D, L-Glu (10 mM, 5 nl) wasmicroinjected bilaterally during the brief (500 ms) pulse; the moments of injections are indicated by brown arrows. Traces for TI,TE, and fR, represent corresponding running time intervals of these parameters before, during, and recovery from L-Glumicroinjection.



s; Fig. 3D), documenting site-specificity of the pertur-bations.

Perturbations of respiratory rhythm and pattern bymicroinjection of gabazine and strychnine in thepre-BötC in vivo and in situMicroinjections of the gabazine-strychnine cocktail wereperformed after initially identifying the pre-BötC regionelectrophysiologically by recording pre-I/I neuronal pop-ulation activity and/or also after microinjected L-Glu(in vivo) to identify the pre-BötC region (Fig. 3C). Pertur-bations of neural activity with bilateral microinjections of110 nl of 250 �M of gabazine and strychnine in the anes-thetized adult rat in vivo were stereotypical. A represen-tative example is shown in Fig. 4A. Drug microinjectionsin pre-BötC produced an increase in the respiratory fre-quency (fR), primarily due to shortening of TE, as well as adecrease of the amplitude of integrated PN activity. Thegroup (n � 6) in vivo data are summarized in Figures4B–E, which shows the mean time courses of the devel-oping perturbations of all parameters (normalized tocontrol values) during and after the microinjections. Per-turbations developed rapidly, within 10 s following theonset of microinjection in all cases. The respiratory fre-quency increased to 184.8 � 5.6% (p � 0.03), TI de-creased to 71.3 � 4.5% (p � 0.03), and TE was reducedto 52.6 � 3.2% (p � 0.03) of control values. The amplitudeof integrated PN activity decreased to 32.1 � 3.9% (p �0.03) of pre-injection control values. These parametervalues, all of which are statistically significant, were ob-tained at 95.83 � 10.03 s after onset of the microinjec-tions when the perturbations approached maximumvalues. The average injection time for the group was57.66 � 22.09 s. After these perturbations, the time re-quired for recovery of inspiratory activity with a patternresembling the control activity was variable, but usually aperiod of minimally 40 min was required in vivo.

Similarly rapid and large perturbations were caused bybilateral microinjections of 30 �M gabazine-strychninewithin the pre-BötC of in situ perfused juvenile rat brain-stem–spinal cord preparations (n � 6; Fig. 5A,B). Figure5A shows a typical example of the large disturbances ofthe respiratory frequency and amplitudes of activity re-corded from PN and cVN, including the pronounced in-crease in the frequency of PN inspiratory dischargeaccompanied by reductions in discharge amplitude. In theexample shown in Figure 5B, the augmented frequencyof PN inspiratory discharge culminated in tonic activity,which occurred in one of six experiments. For the groupdata mean values of respiratory frequency increased to175.9 � 18.0% (p � 0.03), TI slightly increased to 110.4 �4.0% (p � 0.03), and TE decreased to 43.7 � 8.0% (p �0.03) of control values (Fig. 6A–C). The integrated PNactivity amplitude decreased to 62.3 � 6.0% (p � 0.03;Fig. 6D). These disturbances followed a similar timecourse to those in vivo and were measured at 84.1 � 17s after the onset of microinjections (average injection timeof 178.3 s) when maximum changes in parameter valuesobtained. Recovery of control patterns of nerve activity

typically occurred 15–20 min after terminating the bilateralmicroinjections in these in situ experiments.

Disruption of respiratory rhythm and pattern bymicroinjection of gabazine and strychnine in theBötC in vivo and in situBilateral microinjections of gabazine-strychnine were per-formed in the BötC region after identifying this region byextracellular recording of augmenting expiratory and/orpost-I neuronal population activity in situ and in vivo, andin some (n � 3/6) of the in vivo experiments in this group,after confirming the characteristic suppression of re-corded phrenic discharge and pressor blood pressureresponses by L-Glu microinjections. In contrast to theresults obtained with microinjections in the pre-BötC,bilateral microinjections of 110 nl of 250 �M gabazine andstrychnine into the BötC in vivo (Fig. 7; n � 6), and alsothe microinjections in the in situ experiments with thelower concentrations of the inhibitory receptor antago-nists (Figs. 8, 9; n � 6), significantly reduced the fre-quency of integrated PN discharge due primarily to aprolongation of TE, accompanied by a reduction of TI andthe amplitude of integrated PN activity. For the averagedgroup in vivo data (Fig. 7B–E), at 36.66 � 6.4 s followingthe onset of microinjections, the respiratory frequencydecreased to 25.8 � 8.0% (p � 0.03), TI was reduced to88.4 � 5.3% (p � 0.06), and TE increased to 257.2 �48.0% (p � 0.03) of control values. The amplitude ofintegrated phrenic discharge decreased to 39.9 � 13.8%(p � 0.03) of control values (Fig. 7E). In two in vivoexperiments, rhythmic inspiratory discharge was com-pletely suppressed for �20 s (Fig. 7A). The average mi-croinjection time for the group was 53.67 � 19.32 s.

Similar perturbations occurred with bilateral microinjec-tions of 30 �M gabazine-strychnine in the BötC in situ(Figs. 8, 9). For the averaged group data (n � 6, Fig.9A–C) at 92.5 � 16.77 s after injection onset, inspiratoryfrequency decreased to 22.6 � 10.7% (p � 0.03), TE

increased to 430.2 � 17.3% (p � 0.03), and TI decreasedto 56.2 � 13.0%, (p � 0.03) of pre-injection controlvalues. Integrated PN discharge amplitude was reducedto 15.4 � 7.0% (p � 0.03; Fig. 9D). In three of these in situexperiments, rhythmic inspiratory activity was transientlysuppressed for �20 s (Fig. 8). These perturbations werereflected in simultaneously recorded cVN inspiratory ac-tivity (Fig. 8A) in all experiments analyzed and by re-corded pre-BötC pre-I/I population activity (Fig. 8B) intwo of these experiments, which demonstrate that theperturbations in BötC disrupt pre-BötC neuronal activity.In all experiments post-I cVN discharge was also dis-rupted (below) and in one experiment tonic discharge onthe cVN was recorded during the disruption of rhythmicmotor output (Fig. 8A). With the bilateral injections of theinhibitory receptor antagonists in the BötC in situ or invivo, although nerve activity could emerge after transientsuppression of motor output (Figs. 7A, 8A), such activitywas disturbed relative to control activity. In all of theseexperiments, typically 15–25 min in situ and 40–60 min invivo were required to recover activity resembling controlpatterns of motoneuronal discharge.



Disruption of three-phase respiratory pattern byblocking inhibition in the pre-BötC or BötC in situThe microinjections of gabazine-strychnine in either thepre-BötC (n � 6) or BötC (n � 6) in situ disrupted the

three-phase respiratory motor output pattern as analyzedfrom simultaneous recordings of PN and cVN activity, thelatter of which always exhibited prominent post-I dis-charge. Cycle-triggered averages and time-series raster

Figure 4. Perturbations of respiratory activity by pharmacologically disrupting GABAAergic and glycinergic inhibition in the pre-BötC ofanesthetized adult rat in vivo. A, Simultaneous bilateral microinjections of gabazine and strychnine (both 250 �M delivered by slowmicroinjections of 110 nl during the time interval indicated by the brown bar at the top and blue rectangle) caused an increase of respiratoryfrequency and a reduction in the integrated phrenic nerve activity amplitude (�PN; black trace, top). The increase of respiratory frequency(fR; green trace, bottom) was mainly due to reductions of expiratory phase duration (TE, blue trace) accompanied by only a small increaseof inspiratory phase duration (TI, red trace). B–E, Group mean time series showing developing changes of normalized TI (B, red curve), TE(C, blue curve), fR (D, green curve), and �PN amp (E, magenta curve) computed over the time window shown from the start (time � 0) ofmicroinjection. Data were computed from �PN for this representative experimental group (n � 6). Solid colored curves are group mean timeand mean normalized parameter values; gray shaded bands are �1 SEM for the mean parameter values. Endpoints shown are mean timeand normalized parameter values �1 SEM for both at the maximal perturbation for the injection periods used.



Figure 5. Perturbations of respiratory activity by pharmacologically disrupting GABAAergic and glycinergic inhibition in the pre-BötCof juvenile rat perfused brainstem–spinal cord in situ. A, Example of experimental recordings illustrating perturbations of integratedPN (�PN) and cVN (�cVN) activities by simultaneous bilateral microinjections of gabazine and strychnine (both 30 �M; injection periodindicated by the brown bar at the top and blue rectangle), which caused an increase of respiratory frequency (fR) due to a reductionin TE (blue) without significant changes in TI, and a reduction in amplitude of �PN and �cVN. B, Example of perturbations where the �PNactivity progressed to tonic activity, as indicated by the upward shift of the �PN signal baseline. Analysis of respiratory parameters in thiscase was performed up to the time point indicated by the vertical dot-dashed line. The progressive reduction in �cVN amplitude in bothexamples reflects in part a reduction and eventual loss of post-I activity (Fig. 10 shows a more detailed analysis).



plots of integrated PN and cVN shown in Figure 10A,Billustrate that cVN post-I activity is eliminated asGABAAergic and glycinergic inhibition is attenuated ineither region. This loss of post-I activity as the drug-induced perturbation develops represents a transforma-tion from a three-phase to a two-phase motor outputpattern (Smith et al., 2007).

Site-specific perturbations of rhythm and pattern bymicroinjections of muscimol in the pre-BötC andBötC in vivo and in situThe differential perturbations of inspiratory discharge fre-quency with the GABAA and glycine receptor antagonistsin the BötC (frequency decrease) versus the pre-BötC(frequency increase) demonstrate site-specificity of theperturbations. To further test for site-specific perturba-tions by manipulating local inhibition, we regionallyinhibited/attenuated neuronal activity by bilateral micro-injection of the GABAA receptor agonist muscimol. Inthese experiments, the pre-BötC and BötC regions werefirst identified electrophysiologically by extracellular re-cording and/or by microinjection of L-Glu in vivo. As illus-trated in Figure 11, bilateral microinjection of 25–30 nl of100 �M muscimol in the pre-BötC in vivo progressivelyreduced the frequency and amplitude of phrenic dis-charge and eliminated inspiratory motor output within 20s in all experiments (n � 7). The suppression of inspiratory

activity persisted for 40.1 � 26.8 s during which rhythmicinspiratory activity could be restored immediately by sub-sequent bilateral microinjection of 110 nl of 250 �M gaba-zine at the same site (Fig. 11). This latter result alsodemonstrated the efficacy of gabazine at the concen-trations used in our experiments to antagonize activa-tion of GABAA receptors in a regionally specific manner.Bilateral microinjections of lower concentrations ofmuscimol (10 �M) in the pre-BötC in the in situ prepa-rations (n � 5) also rapidly suppressed phrenic inspira-tory activity (Fig. 12).

In experiments targeting the BötC in vivo (n � 7), bilat-eral microinjection of the same volume and concentrationof muscimol progressively increased discharge fre-quency, due primarily to a reduction of TE, and reducedthe amplitude of integrated phrenic inspiratory discharge(Fig. 13). For the averaged group in vivo data at 486.4 �67.6 s after injection onset, integrated phrenic dischargeamplitude was reduced to 58.9 � 8.9% (p � 0.015),inspiratory frequency increased to 175.19 � 8.4%(p � 0.015), TE decreased to 51.4 � 3.3% (p � 0.015), andTI decreased to 83.4 � 3.3% (p � 0.015) of pre-injectioncontrol values (Fig. 13B–D). Comparable perturbationswere obtained with bilateral microinjections of 10�M mus-cimol in the in situ preparations (Fig. 14; n � 6). At 139 �20 s following the onset of the microinjections, the groupaveraged inspiratory frequency increased to 148.1 �

Figure 6. Group data summarizing changes of respiratory activity parameters by pharmacologically disrupting GABAAergic andglycinergic inhibition in the pre-BötC of juvenile rat perfused brainstem–spinal cord in situ. A–D, Mean time series from the start ofmicroinjection showing developing changes of normalized TI (A, red curve), TE (B, blue curve), respiratory frequency (fR; C, greencurve), and integrated PN discharge amplitude (D, amp, magenta curve), which were computed from integrated phrenic nerve activityfor a representative experimental group (n � 6). Solid colored curves are group mean time and normalized parameter values; graybands are �1 SEM for the mean normalized parameter values as in Figure 4B–E.



16.88% (p � 0.03), TE decreased to 53.1 � 8.0% (p �0.03), and TI was essentially unchanged (99.0 � 15.3% ofpre-injection values, p � 1.0; Fig. 14D–F). The integratedphrenic discharge amplitude was reduced to 75.4 � 6.4%(p � 0.03; Fig. 14G). In addition, integrated cVN post-Iactivity was progressively reduced and eliminated bymuscimol in the BötC in situ (Fig. 14B).

Discussion

Role of synaptic inhibition within and between pre-BötC and BötC for respiratory rhythm and patterngenerationFor the discussion of the role of synaptic inhibition inrespiratory rhythm and pattern generation, it is necessary

Figure 7. Perturbations of respiratory activity by pharmacologically disrupting of GABAAergic and glycinergic inhibition in the BötCof adult rat in vivo. A, Gabazine and strychnine cocktail (110 nl, 250 �M) slowly injected during time period indicated (by the brownbar at the top and blue rectangle), progressively reduced respiratory frequency leading to transient apnea in this example (�PN, blacktrace, top). Changes in the inspiratory (TI, red) and expiratory (TE, blue) phase durations and respiratory frequency (fR) are shown atthe bottom; fR was reduced mainly due to prolongation of TE. B–E, Mean time series from the start of microinjection showingdeveloping changes of normalized TI (B, red curve), TE (C, blue curve), fR (D, green curve), and integrated PN activity amplitude(E, magenta curve) for a representative experimental group (n � 6). Solid colored curves are group mean time and normalizedparameter values; gray bands are �1 SEM for the mean normalized parameter values.



Figure 8. Disruption of rhythmic respiratory activity by block/attenuation of GABAAergic and glycinergic inhibition in the BötC of in situperfused brainstem–spinal cord preparations. A, B, Two examples from experiments using different preparations illustratingperturbations caused by microinjections of gabazine and strychnine (30 �M slowly injected during the period indicated by the brownbars at the top and blue rectangles). As in the vivo experiments, respiratory frequency was reduced by a prolonged TE, integratedphrenic nerve discharge amplitude (�PN) was also reduced, and ultimately apnea occurred. Simultaneously recorded integrated cVN(�cVN) in A shows disruption of rhythmic activity and tonic discharge (shift of integrated activity baseline) during apneic period.B, Simultaneously recorded integrated pre-BötC pre-I/I population activity from another experiment also reflects the reduction ofinspiratory frequency and termination of rhythmic activity during bilateral microinjections of the inhibitory receptor blockers in BötC.



to clarify our definitions of the terms “rhythm” and “pat-tern”. We define rhythm as the respiratory cycle period/frequency. Respiratory pattern is defined as all features ofactivity occurring within the respiratory cycle, includingthe presence and coordination of the different activityphases (inspiratory, post-inspiratory, late expiratory) asrecorded from neuronal populations/nerve motor outputactivity, as well as the durations, amplitudes, and shapesof integrated activity within phases.

Brainstem respiratory networks consist of excitatoryand inhibitory circuits distributed within interacting re-gions of the medulla and pons including the medullarypre-BötC and BötC, which are proposed to contain corecircuits involved in respiratory rhythm and pattern gener-ation. Although there is general agreement that excitatorypre-BötC circuits generate rhythmic inspiratory activitytransmitted to and driving spinal and cranial inspiratorymotor outputs, functional roles of inhibitory pre-BötC andBötC circuits continue to be debated (Smith et al., 2009,2013, cf. Feldman et al., 2013; Janczewski et al., 2013;Richter and Smith, 2014). It has been postulated thatphasic synaptic inhibition originating in these circuits,known to contain populations of rhythmically activeglycinergic and GABAergic neurons, are critically in-volved in generating the three-phase pattern of respi-ratory neuron activity during normal breathing byshaping firing patterns of active populations of neurons,orchestrating phase transitions, and controlling which

populations are inactive during each phase (Rybaket al., 2004, 2007; Smith et al., 2007, 2009,2013). In-spiratory and expiratory neurons in these regions re-ceive phasic volleys of inhibitory post-synapticpotentials/currents as clearly established by intracellu-lar recordings (Schmid et al., 1996; Molkov et al., 2012;Shevtsova et al., 2014; Richter and Smith, 2014), andthe inhibitory neurons in the pre-BötC and BötC areproposed to interact during the respiratory cycle viamutual inhibitory connections for dynamic control ofrhythm generation, although these interactions havenot been definitely established experimentally.

We have further evaluated roles of the glycinergic andGABAergic synaptic inhibition within the pre-BötC andBötC by regionally disrupting/attenuating this inhibitionpharmacologically with specific receptor antagonists,which has been attempted in previous studies in severalspecies (cat, rat, rabbit) in vivo, most recently in adultrats with conflicting results (Janczewski et al., 2013). Inthe present experiments we have established that phar-macologically attenuating GABAA and glycine receptor-mediated inhibition in the pre-BötC or BötC causes majorsite-specific perturbations of respiratory rhythm and pat-tern both in anesthetized, vagotomized adult rats in vivoand unanaesthetized, vagotomized juvenile rat perfusedbrainstem–spinal cord preparations in situ. The resultsobtained in vivo and in situ are congruent and confirmthat:

Figure 9. Group data (n � 6) summarizing perturbations of respiratory rhythm and motor output pattern parameters by disruptingGABAAergic and glycinergic inhibition in the BötC of in situ perfused brainstem–spinal cord preparations. Solid colored curves in thesetime series are mean time and normalized parameter values and gray bands are �1 SEM for the normalized values of TI (A), TE (B),respiratory frequency (fR; C), and integrated inspiratory activity amplitude (D) computed from the start of microinjections fromrecordings of integrated PN activity.



(1) Blocking inhibition in the pre-BötC augmented in-spiratory discharge frequency due to a shortening of TE,reduced inspiratory discharge amplitude, and eliminatedpost-I activity thereby disrupting the normal three-phaserespiratory pattern. In some cases, rhythmic motor outputwas completely terminated.

(2) Blocking inhibition in the BötC reduced inspiratorydischarge frequency associated with a lengthened TE,reduced the amplitude of inspiratory discharge, disruptedthe three-phase pattern due to a loss of rhythmic post-Iactivity, and in some cases transiently terminated inspira-tory motor output accompanying a loss of pre-BötC in-spiratory activity.

Therefore, we conclude that ongoing glycinergic andGABAAergic inhibition in pre-BötC and BötC circuits arefundamentally involved in dynamical regulation of respi-ratory rhythm generation and are required for generatingthe normal three-phase respiratory pattern. Our studiesalso confirm a fundamental role of BötC inhibitory circuitsand their interactions with pre-BötC inspiratory rhythmgenerating circuits.

Comparisons with results from previous targetedpharmacology studiesOur results, obtained in juvenile and adult rats, areconsistent with previous studies in anesthetized, non-

Figure 10. Disturbances of three-phase respiratory pattern including disruption of post-I activity by bilaterally microinjected gabazineand strychnine (30 �M each) in the pre-BötC (A) or BötC (B) of in situ perfused juvenile rat brainstem–spinal cord preparations. Uppertraces in A and B show seven consecutive overlaid and aligned integrated PN (red traces) and cVN (blue traces) activity signals in thepre-microinjection control period (light red and light blue larger amplitude traces, respectively) and also seven overlaid traces ofrecorded signals during microinjections (dark red and dark blue traces with reduced amplitudes). Simultaneously recorded pre-BötCpre-I/I population activity before and during microinjections (light and dark green traces, respectively) is also shown at the top in Bto indicate activity perturbations in this region occurring with disruption of inhibition in BötC. Signals are aligned (cycle-triggered) atthe onset of PN inspiratory activity indicated by the vertical dotted line in A and B. Raster plots below overlaid traces showconsecutive series of inspiratory-onset aligned respiratory cycles with integrated PN inspiratory (red) and cVN including post-I (blue)activities before and during microinjections. Arrows on the raster plots indicate time interval over which the overlaid traces above theraster plots were obtained for the pre-microinjection period. During microinjections in the pre-BötC or BötC, the amplitude ofinspiratory and post-I activity is progressively reduced, and post-I discharge is eliminated as seen on the aligned (darker) �cVN tracesabove the raster plots and in the raster plots with loss of post-I discharge indicated (white arrow).



vagotomized adult cats (Pierrefiche et al., 1998) demon-strating that bilateral pharmacological disruption ofglycinergic synaptic inhibition with strychnine in the pre-BötC reduces inspiratory discharge amplitude and mark-edly augments inspiratory discharge frequency or totallyabolishes rhythmic phrenic nerve activity. In their studies,simultaneous block of GABAAergic and glycinergic inhibi-tion led to sustained tonic discharge on phrenic nerves,indicating disruption of rhythmic inspiratory motor output.Furthermore, results obtained in anesthetized vagoto-mized adult rabbits by Bongianni et al. (2010) also dem-onstrated that bilateral block/attenuation of glycinergicinhibition in the pre-BötC augmented inspiratory fre-quency and reduced inspiratory discharge amplitude.Also, blocking GABAergic inhibition with gabazine in theBötC strongly depressed inspiratory amplitude and fre-quency leading to apnea as we demonstrated withGABAAergic and glycinergic antagonists in several cases.Moreover, in these studies in rabbits, bilateral microinjec-tion of muscimol in the BötC potently augmented inspira-tory discharge frequency and caused loss of rhythmicinspiratory activity that was replaced by tonic discharge ofphrenic nerves.

The general conclusions from these previous studiesin both vagotomized and vagus-intact animals, similarto our conclusions, are that ongoing neuronal GABAA

and glycine receptor-mediated postsynaptic inhibitionin the pre-BötC and BötC have a major role in control-ling the frequency and amplitude of inspiratory circuitactivity during eupneic breathing in vivo. Other results

obtained by pharmacologically blocking glycinergicinhibition with systemically applied strychnine in per-fused rat and mouse brainstem–spinal cord prepara-tions in situ, disturbing synaptic inhibition throughoutthe respiratory network, also demonstrate major distur-bances of respiratory rhythm and disruption of thethree-phase respiratory pattern at cellular and circuitlevels (Shevtsova et al., 2011, 2014; Richter and Smith,2014).

All of the above results and conclusions contrast withthose from the recent pharmacological study by Jancze-wski et al. (2013) performed in anesthetized spontane-ously breathing, vagus-intact or vagotomized adult rats invivo. They reported that sequential bilateral microinjec-tions of bicuculline and strychnine in the pre-BötC andthen BötC via a ventral surface approach does not signif-icantly disturb inspiratory frequency in vagotomized rats,whereas in vagus-intact rats inspiratory frequency wassignificantly reduced, accompanied by a prolongation ofTI and TE– perturbations that the authors attributed to onlysuppression of the Breuer–Hering inspiratory inhibitoryreflex (BHIR), supported by their results showing that theBHIR is blocked by antagonizing synaptic inhibition in thepre-BötC. Other results presented suggested that blockof inhibition in the pre-BötC and BötC does not disturbgeneration of laryngeal post-I activity in vagus-intact ratsand thus they concluded that inhibitory circuit interactionsdo not participate in normal three-phase respiratory pat-tern generation. This result conflicts with our present andprevious results (Shevtsova et al., 2011) in situ, where we

Figure 11. Suppression of rhythmic inspiratory activity by bilateral microinjection of muscimol in the pre-BötC of adult rat in vivo andgabazine antagonism of GABAA receptor activation. Muscimol (30 nl, 100 �M) microinjection (during yellow shaded area) rapidlyreduced integrated PN activity and produced a long-lasting suppression of inspiratory activity that could be restored by bilateralmicroinjection of gabazine (110 nl, 250 �M) at the same site in the pre-BötC (blue shaded area). �PN, phrenic nerve integrated activity;fR, respiratory frequency.



could routinely record post-I activity on cVN or post-Ineuronal activity in this unanaesthetized preparation, andfound that disruption of synaptic inhibition in the pre-BötCor BötC consistently eliminated post-I activity. Anothermajor result by Janczewski et al. (2013) was that bilateralablation of the BötC did not significantly perturb inspira-tory frequency or inspiratory–expiratory phase durations,from which they concluded that the BötC is not involvedat all in generating the three-phase rhythmic respiratorypattern. This clearly contrasts with our results and thoseof Bongianni et al. (2010) that disrupting inhibition in theBötC can cause apnea, whereas suppressing BötC neuro-nal activity with muscimol augments inspiratory frequencyand can lead to apneustic-like tonic discharge, similar todisrupting inhibition in the pre-BötC. Janczewski et al. (2013)presented data showing a similar augmentation of frequencyleading to tonic discharge and disruption of rhythm gener-ation with muscimol injections in the pre-BötC, which is theopposite of ours and previous results that suppressingneuronal activity with muscimol in the pre-BötC potentlyreduces inspiratory frequency and rapidly terminates in-spiratory rhythm generation. Overall Janczewski et al. (2013)concluded that inhibition is not required for a normal eupneicbreathing rhythm in vivo except for mediation of the BHIR invagus-intact animals.

These major discrepancies are not readily resolved. Theresults obtained with these targeted pharmacological ap-proaches depend critically on accurate site-directed de-livery of the inhibitory agonists/antagonists, the ability toantagonize inhibitory postsynaptic receptors on a suffi-

cient number of neurons by regional spread of the antag-onists/agonists to ultimately perturb motor outputs, andalso on regionally confining the pharmacological pertur-bations. In our experiments, we verified locations of injec-tion sites histologically and we consistently obtaineddifferential, regionally specific perturbations of inspiratoryrhythm: slowing of the rhythm in all cases by inhibitoryantagonists in BötC, and contrasting augmented inspira-tory frequency with antagonists in pre-BötC. Our recon-structed microinjection sites appear to be identical to thesites reconstructed by Janczewski et al. (2013, their Fig.1; compare to our Fig. 1). In our in vivo experiments usinga similar ventral surface approach in adult anesthetizedrats, we used identical concentrations and ejection vol-umes of antagonists, which we also showed, at least forgabazine, had pharmacologically effective GABAA receptorantagonist-agonist interactions (Fig. 11). With our targetingprocedures, which included mapping of characteristic neu-ronal activity profiles, as well as differential respiratory andblood pressure responses to local L-Glu microinjections, wealso consistently obtained regionally specific perturbationswith muscimol microinjections at targeted sites: reducedinspiratory frequency and apnea in the pre-BötC, but aug-mented inspiratory frequency in the BötC. This specificityalso suggests that it was possible to confine actions of theagonists/antagonists to the targeted region. Differences inthe numbers of neurons and inhibitory synapses affected bylocal diffusion of the antagonists might contribute, althoughassuming accuracy of targeting, we would expect similar

Figure 12. Illustration of prolonged suppression of rhythmic inspiratory activity by bilateral microinjection of muscimol (10 �M) in thepre-BötC (yellow shaded area) of juvenile rat perfused brainstem–spinal cord in situ. As in vivo, muscimol rapidly reduces phrenic nerveinspiratory discharge frequency and terminates inspiratory activity. �PN, phrenic nerve integrated activity; fR, respiratory frequency.



perturbations because ejection volumes and antagonistconcentrations used in vivo were identical.

Relation to studies using selective targeting ofinhibitory neuronsIn a recent study using optogenetic approaches to selec-tively stimulate or inhibit pre-BötC glycinergic neurons by

virally-transduced expression of photosensitive opsinschannelrhodopsin (ChR2) or archaerhodopsin in sponta-neously breathing adult mice in vivo, Sherman et al. (2015)found that optical stimulation of this subset of inhibitoryneurons in pre-BötC in vivo can terminate inspiration,delay the onset of inspiration with photoactivation duringthe expiratory phase, and produce long-lasting apnea

Figure 13. Perturbation of inspiratory activity by bilateral microinjection of muscimol in the BötC of anesthetized adult rat in vivo. A, Muscimol (30nl, 100 �M) slowly microinjected (brown bar at the top and yellow rectangle) in the BötC augments PN frequency due to a reduction of TE. B–E,Mean time series for group data (n � 7) summarizing perturbations of respiratory rhythm and motor output pattern parameters. Solid coloredcurves are mean time and normalized parameter values and gray bands are �1 SEM of TI (B), TE (C), respiratory frequency (fR; D), and integratedinspiratory activity amplitude (E) computed from recordings of integrated phrenic nerve activity as in previous figures.



Figure 14. Disturbances of respiratory pattern including elimination of post-inspiratory activity by bilateral microinjection of muscimol(10 �M) in the BötC of juvenile rat perfused brainstem–spinal cord in situ. A, Representative example of augmented �PN inspiratorydischarge frequency and pronounced reduction of cVN integrated (�cVN) activity amplitude due to suppression of post-I activity.Overlaid traces in B and time-series raster plot in C of consecutive respiratory cycles aligned to inspiratory onset (indicated by verticaldotted line in B) illustrate perturbations of cVN post-I discharge during BötC muscimol microinjection in another experiment. B,Integrated PN (red traces) and cVN (blue traces) activity are shown before (lighter red and lighter blue traces, respectively) and after(dark red and dark blue traces) muscimol microinjection. Aligned traces of �PN (red) and �cVN (blue) are shown in raster plot in C withthe loss of post-I cVN discharge indicated (white arrow), which is also clearly seen from the �cVN traces (dark blue) above. D–G, Meantime series for group data (n � 6) summarizing perturbations of respiratory rhythm/pattern parameters. Solid colored curves are meantime and parameter values and gray bands are �1 SEM of normalized TI (D), TE (E), inspiratory frequency (fR; F), and integratedinspiratory activity amplitude (G) computed from recordings of integrated PN activity.



with prolonged photostimulation. Conversely, prolongedphotoinhibition augmented the amplitude and frequencyof inspiratory activity and could reverse reflex-inducedapneas. This approach can provide important informationon dynamic perturbations resulting from augmentationor loss of inhibitory neuron function (Abdala et al., 2015)and their results demonstrate that at least pre-BötCglycinergic neuron activity can strongly modulate on-going inspiratory rhythm and pattern generation. Theauthors concluded, however, that glycinergic inhibitionis not essentially involved in rhythmogenesis despitethese perturbations because inspiratory rhythm per-sisted after photoinhibition, although as the authorsdiscuss, it is unclear whether sufficient numbers ofglycinergic neurons were optically silenced to reveal fullaffects of pre-BötC glycinergic neuron activity onrhythm generation.

Another important consideration for interpreting thesignificance of these results is that silencing pre-BötCglycinergic neurons may not be analogous to pharmaco-logically blocking even postsynaptic glycinergic recep-tors, particularly because relevant glycinergic synapsesmay originate from neurons outside of the pre-BötC suchas BötC neurons not transduced in sufficient numberswith viral vectors targeted to the pre-BötC. These opto-genetic results also cannot be readily compared with theeffects of pharmacological blockade of both glycinergicand GABAAergic postsynaptic inhibition. As known, pre-BötC contains inhibitory neurons with each type of trans-mission, as well as neurons coexpressing both GABA andglycine (Schreihofer et al., 1999; Koizumi et al., 2013), soall these neuron types would have to be optically inhibitedto fully evaluate the functional role of local inhibitory cir-cuits in the pre-BötC. The augmented inspiratory fre-quency that we observed with disrupting inhibition inthe pre-BötC, which has also been observed by phar-macologically attenuating only glycinergic inhibitionwith strychnine (Pierrefiche et al., 1998) as discussedabove, is a common result of both pharmacological andoptical approaches. However, inspiratory amplitudewas always markedly reduced, not augmented, bypharmacologically perturbing postsynaptic inhibition inthe pre-BötC, suggesting that more complex inhibitoryinteractions in pre-BötC inspiratory rhythm generationcircuits are revealed by the pharmacological ap-proaches. Experiments were not presented by Shermanet al. (2015) on photostimulation/inhibition of glyciner-gic neurons in the BötC. Optical stimulation of BötCneurons transduced with ChR2, although not selec-tively in GABAergic and glycinergic neurons but likelyincluding these neurons in these studies, strongly sup-presses inspiratory activity in contrast to the powerfulaugmentation of frequency with photostimulation ofpre-BötC neurons in anesthetized rats in vivo (Alsahafiet al., 2015). These observations are analogous to ourresults with local glutamate microinjections, which alsodo not selectively stimulate specific neuronal pheno-types in these regions.

Normal eupneic rhythm generation in the absence ofsynaptic inhibition in vivo?A continuing debate is whether a normal eupneic inspira-tory rhythm and respiratory pattern can be generatedwithout synaptic inhibition in the pre-BötC in the intactsystem where the pre-BötC interacts with the BötC andother sources of phasic/tonic synaptic inhibition. Weshow that disrupting pre-BötC synaptic inhibition leadseither to higher frequency oscillations accompanied by alarge reduction in the amplitude of pre-BötC/phrenic in-spiratory activity or to tonic phrenic nerve activity in somecauses, indicating that a normal eupneic inspiratoryrhythm generally does not occur in the absence of syn-aptic inhibition. The variable persistence of inspiratoryrhythmic activity in other cases, albeit with large distur-bances of the inspiratory rhythm and inspiratory–expira-tory pattern, indicates that either we did not sufficientlyblock postsynaptic inhibition in the pre-BötC with ourtargeted pharmacological approach in these cases, orsome form of inspiratory rhythm generation occurs afterdisruption of GABAAergic and glycinergic inhibition in theintact system. The former is a technical problem that isdifficult to solve. This would require extensive local intra-cellular recordings to analyze endogenous postsynapticinhibitory potentials simultaneously with the pharmaco-logical perturbations. In our approach, we limited themicroinjection volumes of the pipette solution containinginhibitory antagonists, in an attempt to achieve site-specificity of the perturbations. This may have precludedsufficient inhibitory receptor block throughout the localcircuits to cause complete disruption of inspiratoryrhythm generation in these cases. On the other hand,pre-BötC excitatory neurons and circuits have intrinsicrhythmogenic properties (Smith et al., 2007; St. Johnet al., 2009; Richter and Smith, 2014), so it is possible thatsome form of inspiratory rhythm generation, albeit abnor-mal, can persist in the absence of synaptic inhibition in theintact juvenile/adult system, as occurs in the neonatalrat/mouse pre-BötC isolated in slices in vitro.

Our models of excitatory and inhibitory pre-BötC cir-cuits and their interactions with BötC inhibitory neurons inthe intact system incorporate intrinsic rhythmogenic prop-erties in the excitatory pre-BötC population (Rybak et al.,2004, 2007; Smith et al., 2007; Rubin et al., 2009). Thesemodels indicate that depending on the initial excitationstate of the excitatory kernel neurons, the endpoint aftercomplete block of postsynaptic inhibition can be eithertonic activity of pre-BötC neurons at high levels of exci-tation or rhythmic activity at lower excitation levels. It isclear that to answer the question of whether any form ofinspiratory rhythm generation can occur, intracellular re-cordings will have to be used to monitor the level ofmembrane potential and activity patterns during and afterlocal block of postsynaptic inhibition.

Disorganization of the three-phase respiratorypattern after disrupting postsynaptic inhibitionThe three-phase organization of the respiratory pattern atleast in situ was disrupted accompanying the large dis-turbances of inspiratory rhythm by blocking inhibition in



the pre-BötC or BötC. This confirms that the normaleupneic breathing pattern with appropriately coordinatedspinal and cranial motoneuron activity relies on synapticinhibition in BötC and pre-BötC circuits. These inhibitorycircuits normally operate with pontine and other excitatoryinputs (Rybak et al., 2004; Smith et al., 2007; Dutschmannand Dick, 2012). The pontine Kölliker–Fuse nucleus proj-ects heavily to both the pre-BötC and BötC affecting theirinhibitory interactions. Specifically, pontine input is re-quired to generate post-I activity (Rybak et al., 2004;Smith et al., 2007; Dutschmann and Dick, 2012) so theobserved loss of this activity in part reflects disruption ofthe balance of excitatory and inhibitory interactions. Re-cent experiments in a transected perfused rat brainstempreparation have confirmed the critical role of the pons inthree-phase respiratory pattern generation (Jones andDutschmann, 2016). Previously it has been demonstratedthat blocking glycinergic inhibition systemically causes ashift of post-I neuron activity into the inspiratory phase,due to phasic excitation of post-I neurons that is normallyshunted by inspiratory phase inhibition (Richter andSmith, 2014) and this shift causes abnormal glottal con-striction during inspiration (Dutschmann and Paton,2002). This also implies that the shifted post-I inhibitoryneurons would provide abnormally timed inhibition duringthe inspiratory phase, possibly including to inspiratoryneurons in bulbospinal circuits downstream from theBötC and pre-BötC, which could contribute to the largereductions of inspiratory amplitude and attenuation of PNramping inspiratory discharge observed after disruptinginhibition. The loss of the three-phase pattern is predictedfrom models (Rybak et al., 2004, 2007; Smith et al., 2007;Shevtsova et al., 2011, 2014) after inhibitory block in theBötC, where post-I inhibitory neuronal activity is pre-sumed generated. Our results that the three-phase pat-tern is also disrupted after blocking inhibition in thepre-BötC may reflect the known presence of post-Ineurons in the pre-BötC (Schwarzacher et al., 1995;Alheid and McCrimmon, 2008), and shows that thepre-BötC also plays a role in three-phase pattern gen-eration, conceivably by either interactions with theBötC and/or by local inhibitory circuit interactions,which need to be delineated.

Role of BötC circuits and synaptic inhibition inrespiratory pattern generationA fundamental role for BötC neurons in respiratory patterngeneration has been questioned by Janczewski et al.(2013) as noted above. Their conclusion that this regionplays no role is incompatible with the present and previ-ous results showing that augmenting BötC neuron activity(by glutamate microinjections) suppresses pre-BötC in-spiratory activity, whereas suppressing BötC activity (bymuscimol) augments pre-BötC inspiratory rhythm. TheBötC is known to contain major populations of activeexcitatory and inhibitory expiratory post-I and aug-E neu-rons, and this region is critical for generating post-I activ-ity (Burke et al., 2010). Excitatory BötC neurons arethought to be a major source of expiratory activity in therespiratory network including for generation of post-I pre-

motoneuron activity. BötC glycinergic and GABAergic ex-piratory interneurons are proposed to provide widelydistributed network synaptic inhibition during expiration(Jiang and Lipski, 1990; Tian et al., 1999a,b; Ezure et al.,2003a,b). These postulated inhibitory connections includeto pre-BötC excitatory and inhibitory inspiratory neuronsto provide phasic synaptic inhibition orchestrating rhyth-mic alternation between expiratory and inspiratory activityin the network. Mutual inhibitory synaptic interactionsbetween BötC inhibitory (post-I and aug-E) expiratoryneurons and inhibitory inspiratory (e.g., early-I) pre-BötCneurons have been proposed to coordinate generation ofthe three-phase pattern of neuronal activity during eup-neic breathing (Rybak et al., 2004, 2007; Smith et al.,2007; Rubin et al., 2009; Shevtsova et al., 2011, 2014;Richter and Smith, 2014). Based on these proposed in-teractions, augmenting BötC activity or disrupting post-synaptic inhibition within the BötC should suppressrhythmic activity in the pre-BötC, assuming in each casethat BötC neurons shift from phasic to tonic activity.Correspondingly, suppressing BötC activity should aug-ment inspiratory discharge frequency, and depending onthe level of ongoing pre-BötC excitation, this can lead totonic inspiratory activity. Both of the above are features ofthe present experimental results.

We note that the projections and postsynaptic targetsof BötC and pre-BötC inhibitory interneurons, particularlymutual inhibitory connections between these neurons,have not been mapped structurally. Establishing theseconnections remains an important problem. Nevertheless,our results are consistent with mutual interactions and afundamental role of the BötC and its expiratory inhibitoryneurons in three-phase respiratory pattern generation andcontrol of inspiratory rhythm.

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