J Physiol 579.3 (2007) pp 863–876 863

Breathing dysfunctions associated with impaired controlof postinspiratory activity in Mecp2−/y knockout mice

Georg M. Stettner1, Peter Huppke1,2, Cornelia Brendel1, Diethelm W. Richter2,3, Jutta Gartner1,2

and Mathias Dutschmann2,3

1Department of Pediatrics and Pediatric Neurology, Georg August University, Robert-Koch-Str. 40, 37075 Gottingen, Germany2DFG Research Center Molecular Physiology of the Brain (CMPB), Humboldtallee 23, 37073 Gottingen, Germany3Department of Neuro and Sensory Physiology, Georg August University, Humboldtallee 23, 37073 Gottingen, Germany

Rett syndrome (RTT) is an inborn neurodevelopmental disorder caused by mutations in

the X-linked methyl-CpG binding protein 2 gene (MECP2). Besides mental retardation,

most patients suffer from potentially life-threatening breathing arrhythmia. To study its

pathophysiology, we performed comparative analyses of the breathing phenotype of Mecp2−/y

knockout (KO) and C57BL/6J wild-type mice using the perfused working heart–brainstem

preparation (WHBP). We simultaneously recorded phrenic and efferent vagal nerve activities

to analyse the motor pattern of respiration, discriminating between inspiration, postinspiration

and late expiration. Our results revealed respiratory disturbances in KO preparations that were

similar to those reported from in vivo measurements in KO mice and also to those seen in

RTT patients. The main finding was a highly variable postinspiratory activity in KO mice

that correlated closely with breathing arrhythmias leading to repetitive apnoeas even under

undisturbed control conditions. Analysis of the pontine and peripheral sensory regulation

of postinspiratory activity in KO preparations revealed: (i) prolonged apnoeas associated

with enhanced postinspiratory activity after glutamate-induced activation of the pontine

Kolliker-Fuse nucleus; and (ii) prolonged apnoeas and lack of reflex desensitization in response to

repetitive vagal stimulations. We conclude that impaired network and sensory mediated synaptic

control of postinspiration induces severe breathing dysfunctions in Mecp2−/y KO preparations.

As postinspiration is particularly important for the control of laryngeal adductors, the finding

might explain the upper airway-related clinical problems of patients with RTT such as apnoeas,

loss of speech and weak coordination of breathing and swallowing.

(Received 28 August 2006; accepted after revision 2 January 2007; first published online 4 January 2007)

Corresponding author M. Dutschmann: Department of Neuro and Sensory Physiology, Georg August University,

Humboldtallee 23, 37073 Gottingen, Germany. Email: mdutsch@gwdg.de

Rett syndrome (RTT) is an X-linked neurodevelopmentaldisorder associated with breathing abnormalities andsevere mental retardation in females (Hagberg et al. 1983;Hagberg & Hagberg, 1997). Mutations and gene deletionsin the MECP2 gene located on Xq28 have been identifiedas primary causes of the disease (Amir et al. 1999). TheMECP2 gene encodes the methyl-CpG binding protein 2(MeCP2), which acts as a transcriptional repressor. Thetarget genes regulated by MeCP2 are still incompletelyknown and subject to intensive research (for review seeBienvenu & Chelly, 2006).

The clinical course of RTT is characterized by differentstages (Hagberg & Witt Engerstrom, 1986). Towardsthe end of the regression period, which occurs between1 and 3 years of age, most patients develop state-dependent breathing abnormalities. These abnormalities

are included in the clinical diagnostic criteria for RTT(Hagberg et al. 2002). Respiratory disturbances duringwakefulness comprise alternating periods of hyper-ventilation and apneustic, breath-holding frequentlyterminated by Valsalva’s manoeuvres and forced and deepbreathing, as well as apnoeic breathing (e.g. Elian &Rudolf, 1991; Marcus et al. 1994; Julu et al. 2001; Julu& Witt Engerstrom, 2005). Importantly, these respiratoryarrhythmias are seen as a main cause of sudden andunexpected deaths in RTT patients (Kerr et al. 1997).By contrast, breathing during sleep is apparently stable(Southall et al. 1988; Marcus et al. 1994).

In spite of the clinical impact of the respiratorydysfunction in RTT, little is known about the underlyingpathomechanisms. Now, the availability of the Mecp2−/y

knockout (KO) mouse (Chen et al. 2001; Guy et al. 2001;

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864 G. M. Stettner and others J Physiol 579.3

Shahbazian et al. 2002), an animal model for humanRTT, allows analytic studies of respiratory dysfunction.Viemari et al. (2005) showed recently that Mecp2−/y KOmice have an RTT-like respiratory disorder. The primarycause of the breathing abnormalities in mice is as unclearas in patients. There is some support for the hypothesisof cortical dysfunction and thus behaviourally associatedrespiratory dysfunction (Elian & Rudolf, 1991; Marcuset al. 1994); however, others have reported brainstemimmaturity (Julu et al. 2001; Julu & Witt Engerstrom,2005) or disturbance of neuromodulatory regulation ofsynaptic function within the ponto-medullary respiratorynetworks (Viemari et al. 2005).

In the present study we investigated the respiratorymotor pattern of Mecp2−/y KO mice and control C57BL/6Jwild-type (WT) mice using the decerebrated and arteriallyperfused working heart–brainstem preparation (WHBP;Paton, 1996). Because of the decerebration, potentialinfluences of higher brain regions on the in situ respiratorypattern generators of the ponto-medullary brainstem areexcluded. RTT patients also have upper airway-relatedclinical problems, such as loss of speech and impairedswallowing. Thus we were also interested in the pontinecircuits (e.g. Kolliker-Fuse nucleus (KF), see Dutschmann& Herbert, 2006) and sensory inputs (Hering-Breuerreflex (HBR)) that control postinspiratory mediatedactivation of laryngeal adductors. Our results demonstratea significant impairment of the pontine and sensorycontrol of the postinspiratory motor activities in Mecp2−/y

KO mice preparations.

Methods

The experimental procedures were performed inaccordance with European community and NationalInstitutes of Health guidelines for the care and use oflaboratory animals. The ethical committee of the GeorgAugust University Gottingen approved the study.

The developmental course of the breathing dysfunctionin Mecp2−/y KO mice has previously been described byViemari et al. (2005). Within 6 weeks of age, the KO micedevelop a severe RTT-like breathing phenotype. Our studyhas not focused on the development but rather the rangeof respiratory dysfunctions in KO mice. Thus, we decidedto work on adult KO and WT mice at a postnatal age of40 ± 2 days.

Animal breeding and genotyping

Experiments were performed using the mouse model forRTT, strain B6.129P2(C)-Mecp2tm1–1Bird (Guy et al. 2001).The mice were obtained from The Jackson Laboratory(Bar Harbour, ME, USA) and maintained on a C57BL/6Jbackground. Hemizygous mutant Mecp2−/y males were

generated by crossing heterozygous Mecp2+/− females withC57BL/6J WT males. All mice were routinely genotypedin accordance with The Jackson Laboratory genotypingprotocols (http://jaxmice.jax.org/strain/003890.html).For these purposes genomic DNA was isolated frommice tails using the DNeasy Tissue Kit (Qiagen, Hilden,Germany) according to manufacturer’s protocol.

All experiments were performed in hemizygousMecp2−/y KO and C57BL/6J WT male mice. We exclusivelyused Mecp2−/y KO males because heterozygous Mecp2+/−

female mice have an unpredictable and heterogeneousphenotype due to the X-inactivation profile of the X-linkedMECP2 gene.

WHBP

The experiments were performed using the arteriallyperfused WHBP. Mice were anaesthetized deeply ina saturated atmosphere of isoflurane (1-chloro-2,2,2-trifluoroethyl-difluoromethylether; Abbott, Wiesbaden,Germany). Once the animal failed to respond to noxiouspinch to the tail or a hind paw, it was transected belowthe diaphragm, transferred into ice-cooled (5◦C) artificialcerebrospinal fluid (aCSF) gassed with carbogen (95%O2 – 5% CO2), decerebrated at the precollicular leveland cerebellectomised. The lungs were removed. The leftphrenic nerve (PN) was separated and cut at the levelof the diaphragm. The descending aorta was isolatedfrom the ventral surface of the spinal column. Theseinitial procedures took approximately 10 min. The pre-paration was then transferred to a recording chamber.The descending aorta was cannulated and perfused usinga peristaltic pump (Watson-Marlow, Wilmington, MA,USA) with carbogen-gassed aCSF at 31◦C containing Ficoll(1.25%, Sigma-Aldrich, Steinheim, Germany) to maintaincolloidal osmotic pressure. The perfusate contained (mm):NaCl 125, KCl 3, KH2PO4 1.25, CaCl2 2.5, MgSO4 1.25,NaHCO3 25 and d-glucose 10, and 1.25% Ficoll. Theosmolarity of the perfusate was 300 ± 10 mosmol l−1 andthe pH was adjusted to 7.35 ± 0.05 by gassing withcarbogen. The perfusate was filtered and passed throughbubble traps to remove gas bubbles. The perfusate leakingfrom the preparation was collected and recirculated afterreoxygenation. Cardiac activity returned within secondsand rhythmic contractions of respiratory muscles within afew minutes after onset of reperfusion. Respiratory relatedmovements were abolished by vecuronium bromide(0.3 μg ml−1, Inresa, Freiburg i. Br., Germany).

After paralysis, phrenic nerve activity (PNA) wasprimarily used to fine-tune the preparations byadjusting the flow rate (10–22 ml min−1) and perfusionpressure (40–70 mmHg). For measurement of aorticperfusion pressure, a double-lumen catheter was usedand connected to a pressure transducer. During the

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tuning, the flow rates were individually adjusted tofulfil the following criteria: re-appearance of a clearlyidentifiable three-phase respiratory pattern, rhythmicrespiratory motor discharges of at least 60 bursts min−1

and maintenance of the respiratory pattern (althoughdisturbed in KO mice) for at least 30 min before startingany experiments or data analysis. In addition during thetuning phase, the right central vagus nerve (cVN) wasdissected and prepared for recording.

Nerve and cardiac recordings

Respiratory motor nerve activities were recordedsimultaneously from the central ends of the phrenic andvagal nerves in all preparations via suction electrodesconnected to differential amplifiers (Neurolog 100). Nerveactivity was amplified and filtered (8 Hz to 3 kHz;Neurolog modules 104 and 125). All data were digitizedusing a MacLab 8s interface and stored on a computer usingChart software (version 5.2, ADInstruments, Australia).Respiratory motor nerve activity was integrated (timeconstant, 100 ms). The electrocardiogram was recordedsimultaneously with the PNA.

Data analysis

Using the PNA and central vagal nerve activity (cVNA)recordings we analysed the duration of the followingrespiratory parameters: total respiratory cycle length(T tot), inspiration (T i), postinspiration (Tpi) and lateexpiration (T e2). In contrast to in vivo investigations in thecat, PNA recording does not display a clearly identifiablepostinspiratory activity in mice. Therefore we classifiedthe entire expiratory interval as T e (including Tpi andT e2). Tpi and T e2 were identified and measured from thecVNA. The inspiratory cVNA coincided with the PNA.The duration of the expiratory cVNA was defined asTpi, and the phase with absent cVNA in late expirationas T e2. Breathing frequency was indicated by the PNA(bursts min−1). In addition to the duration we analysed thevariance of the respiratory parameters. These parameterswere calculated from 60 consecutive breathing cycles at arepresentative control activity. Furthermore, we measuredthe frequency and duration of spontaneous apnoeas overa period of at least 5 min. We defined an apnoea as theabsence of rhythmic PNA for a period of at least tworespiratory cycles based on the mean T tot calculated from60 consecutive breathing cycles at a representative controlactivity. Because we focused on the respiratory dysfunctionin Mecp2−/y KO mice in the present study, we did notanalyse cardiovascular parameters.

Pontine microinjections

Subsequent pressure microinjections of glutamate(10 mm, 40 nl, Sigma-Aldrich) and pontamine sky blue(40 nl, Sigma-Aldrich) into the area of the left pontineKF were performed with a micromanipulator-drivenmulti-barrelled micropipette (tip diameter, 30–40 μm).The micropipette was placed just caudally to the leftinferior colliculus and 0.6–0.7 mm medial from the lateralmargin of the pons. The depth of the microinjectionwas 1.4–1.6 mm. The injected volumes were measuredby observing the descending drug meniscus in themicropipette through a binocular microscope fitted witha calibrated eyepiece graticule. After identification ofthe effective injection site (e.g. by transient apnoea ortachypnoea following glutamate injection), pontaminesky blue was injected to mark the area of microinjection.The duration of the glutamate-evoked apnoea and theintegral discharge of the postinspiratory cVNA duringthe glutamate-evoked apnoea were analysed. The endof the glutamate-evoked apnoea was defined by thereappearance of the first PNA burst. At the end of eachexperiment, the brainstem was removed and fixed in4% paraformaldehyde (Merck, Darmstadt, Germany)containing 20% sucrose (Merck). For anatomicalverification of the injection sites we cut series of 50 μmthick coronal sections through the pons with a freezingmicrotome (Reichert and Jung, Wetzlar, Germany).Afterwards, the sections were stained with neutral redsolution (Sigma-Aldrich). The locations of micro-injections were documented on semi-schematic drawingsof coronal sections through the dorsolateral pons adaptedfrom Paxinos & Franklin (2001).

Electrical stimulation of vagal afferents

To mimic the HBR the central branch of the left vagal nervewas electrically stimulated through a suction electrode.The indifferent electrode was placed into the cervical tissuenear to the vagal nerve. We analysed the duration of thesensory evoked reflex apnoea as seen in the PNA andthe desensitization of the postinspiratory reflex apnoeain response to repetitive vagal stimulation (15 trials every60 s, 10 s duration of each stimulation trial, 100 μs pulsewidth, 20 Hz frequency, 0.4–4 mV).

Statistical analysis

Analysis was performed with the Statistica 7.1 softwarepackage (StatSoft Inc., Tulsa, OK, USA). All results arepresented as means ± s.e.m. Respiratory parameters ofWT and KO preparations were compared using pairedStudent’s t test in the case of normally distributed data(phase durations, frequency and duration of spontaneousapnoeas, duration of sensory and glutamate-evoked

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apnoea) and using the Mann–Whitney U test in the case ofnon-normally distributed data (integrated postinspiratoryvagal discharge during glutamate-evoked apnoea). Toindicate the scale of phase durations, the variance of phasedurations measured from 60 consecutive breathing cyclesof every preparation (see data analysis) was calculated.The variances of the phase durations in WT and KO micewere compared using the Mann–Whitney U test because ofnon-normal distribution of the data. Correlations betweentwo phase durations measured from 60 consecutivebreathing cycles of every individual preparation (see dataanalysis) were investigated using Pearson’s correlationcoefficient. All data sets were tested for normal distributionprior to the correlation analysis. For comparison of thecorrelation coefficients between the groups of WT andKO mice, the Mann–Whitney U test was used becauseof non-normal data distribution. P < 0.05 was consideredsignificant.

Results

In the present study we analysed and compared therespiratory phenotype of Mecp2−/y KO and their controlC57BL/6J WT male mice using the arterially perfusedWHBP. For our investigations we used 28 KO and 28WT male mice at postnatal day 40 ± 2. Simultaneousrecordings of PNA and cVNA were performed in allpreparations.

Characterization of the respiratory motor pattern

Both WT (n = 28) and KO preparations (n = 28)generated a respiratory activity characterized by anincrementing inspiratory PNA and a biphasic inspiratoryand postinspiratory vagal discharge pattern (Fig. 1A–D).

WT preparations displayed a fast and regular respiratoryrhythm with a PNA burst frequency of 99.1 ± 5.5 min−1

(Figs 1A and 3A). Analysis of the phase durationsas determined from PNA and cVNA revealed thefollowing values: T i, 0.20 ± 0.01 s; Tpi, 0.24 ± 0.02 s;T e2, 0.21 ± 0.01 s; T e, 0.45 ± 0.03 s; T tot, 0.65 ± 0.03 s(Fig. 2A). Overall, there was a regular three-phasedischarge and the phase relations suggested an eupnoeicrespiratory motor pattern.

In KO preparations the mean durations of theinspiratory, postinspiratory and late expiratory phasesshowed highly significant differences while mean T e

(0.50 ± 0.03 s), T tot (0.67 ± 0.04 s) and burst frequency ofPNA (97.4 ± 5.5 min−1) did not differ significantly fromthose in WT preparations. In particular, the mean Tpi wasmarkedly prolonged (0.41 ± 0.03 s, P < 0.001) whereasthe mean inspiratory (0.17 ± 0.01 s, P < 0.05) and lateexpiratory (0.08 ± 0.01 s, P < 0.001) phase durations inKO preparations were shortened (Fig. 2A).

All KO preparations showed pronounced respiratoryarrhythmia (Figs 1C and 3B). The arrhythmia wascharacterized by a highly significant increase in varianceof T tot, Tpi and T i (each P < 0.001) in comparison to WTpreparations (Fig. 2B). During the arrhythmia individualprolonged Tpi values correlated with prolonged T tot values(Fig. 3B). However, T e2 did not affect the respiratoryarrhythmia in KO preparations because there was nodifference in the variance of T e2 when compared to WTcontrols.

In KO and WT preparations, a relationship betweenmean Tpi and T tot values was observed. However,the mean correlation coefficient between Tpi and T tot

measured from 60 consecutive breathing cycles of eachpreparation was significantly higher in KO than in WTmice (r = 0.97 ± 0.01 versus r = 0.66 ± 0.04, P < 0.001).Almost identical correlation coefficients and a similarlevel of significance were calculated for correlationsbetween Tpi and T e. There was no correlation betweenT e2 and T tot values in KO preparations, whereas WTpreparations showed a marked correlation (r = 0.22 ±0.03 versus r = 0.65 ± 0.03, P < 0.001). These resultsindicate that Tpi mainly determines T e and therefore T tot

in KO mice.All WT preparations displayed unprovoked transient

central apnoeas accompanied by tonic vagal post-inspiratory discharge. These apnoeas occurred at afrequency of 0.85 ± 0.10 apnoeas min−1. By contrast, KOpreparations showed such repetitive apnoeas at a signi-ficantly higher frequency (2.45 ± 0.35 apnoeas min−1,P < 0.001, Fig. 4A). The mean duration of spontaneouscentral apnoeas in WT preparations was 2.73 ± 0.18 s,accounting for a 4.24 ± 0.24-fold increase in the mean T tot.In KO preparations, the spontaneous apnoeas lasted for2.13 ± 0.14 s, accounting for a 3.22 ± 0.15-fold increasein the mean T tot (P < 0.05, Fig. 4B).

Pontine glutamate microinjections

We injected glutamate (10 mm, 40 nl) into the area ofthe left KF in 13 WT and 12 KO preparations. Allinjection sites were marked with pontamine sky blue afterglutamate injection using multi-barrelled micropipettes.The histologically verified locations of microinjectionswere documented on semi-schematic drawings of coronalsections through the dorsolateral pons (Fig. 6).

All glutamate injections that triggered a transientpostinspiratory apnoea were clustered in the intermediatepart of KF (KO, n = 10; WT, n = 9; stars in Fig. 6).The evoked apnoeas following glutamate injections ofcomparable volumes into almost identical areas of theintermediate KF (see Fig. 6) were significantly longer in KOwhen compared to WT preparations (17.37 ± 3.32 s versus4.60 ± 0.63 s, P < 0.01, Fig. 5A–C). In all preparations, the

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Figure 1. Recording of the phrenic (PNA) and central vagal nerve activities (cVNA) of representativewild-type (WT) and knockout (KO) preparationsThe figure shows traces of 20 s duration of a WT and KO preparation (A and C) as well as sections with enlargedtime scale of the same preparations to demonstrate differences in the respiratory phase durations (B and D). A andB, display the eupnoeic breathing pattern of a WT mouse with an incrementing discharge of the phrenic nerveduring inspiration and biphasic inspiratory and postinspiratory discharge of the vagal nerve. There is no dischargein both recorded nerves during the late expiratory phase. C and D, illustrate characteristic features of respiratoryabnormalities in a KO preparation. The rhythm is irregular due to a mixture of fast and slow breathing activityand repetitive apnoeas (∗). Please note the variability of vagal postinspiratory discharge in terms of duration andamplitude (+). D, shows a section with enlarged time scale of stable breathing of the same KO preparation as inC. PNA burst frequency is identical to the WT preparation (see B), but inspiratory duration (T i) is slightly shortenedand postinspiratory duration (Tpi) markedly prolonged.

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Figure 2. Bar diagrams illustrating mean phase durations ± S.E.M. (A) and mean variance of phasedurations ± S.E.M. (B) in 28 wild-type (WT) and 28 knockout (KO) preparationsPlease note the logarithmic scale of the y-axis in B. T i, duration of inspiration; Tpi, duration of postinspiration; Te2,duration of late expiration; Te, duration of the entire expiratory interval; T tot, total respiratory cycle length; n.s.,not significant; ∗P < 0.05; ∗∗∗P < 0.001.

glutamate-evoked respiratory arrest was accompanied bya tonic postinspiratory vagal discharge. This tonic vagalactivity was significantly enhanced in KO when comparedto WT preparations as indicated by the integrated vagalactivity during the glutamate-evoked apnoea (47.4 ± 3.3

Figure 3. Sequential plot of duration of postinspiration (Tpi) and total respiratory cycle length (T tot)values (in s) of 60 consecutive respiratory cycles recorded in a wild-type (WT; A) and a knockout (KO)preparation (B)Graphs are representative for the groups of measured WT and KO mice. Tpi and T tot values are regular in the WTmouse but scattered in the KO mouse. Prolonged Tpi values correlate with prolonged T tot values.

versus 12.5 ± 2.8 mV × 100 ms, P < 0.05, Fig. 5A, B andD).

Injections that did not produce postinspiratory apnoeaswere mainly located in the margins of the KF area andresulted in bradypnoea with enhanced postinspiratory

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activity (diamonds in Fig. 6). In a single preparation,glutamate injection into the KF produced a tachypnoeawithout additional postinspiratory modulation (opencircle in Fig. 6A).

Fictive HBR and desensitization in responseto repetitive vagal stimulation

We evoked a fictive HBR by electrical stimulation ofthe central branch of the vagal nerve in nine WT andnine KO mice. When compared to WT controls, thesensory evoked apnoeas in KO preparations were allprolonged and lasted for several seconds after the stimulusoff-switch (Fig. 7A–C). The first stimulation trial revealeda mean duration of reflex apnoea including the 10 svagal stimulation of 19.86 ± 2.43 s in KO preparationswhile the apnoea was significantly shorter in WTpreparations (10.43 ± 0.16 s, P < 0.001, Fig. 7C). All WTpreparations showed a clear desensitization of the HBRin response to repetitive vagal stimulation (15 consecutivestimulation trials every 60 s, 10 s duration of stimulation)with a shortening of the mean evoked reflex apnoea from10.43 ± 0.16 to 3.92 ± 0.50 s (Fig. 7D). By contrast, KOpreparations did not show any sign of desensitization(Fig. 7B and D).

Discussion

Most patients suffering from the inborn neuro-developmental disorder RTT also develop severebreathing arrhythmias. The pathological mechanismsof RTT-associated breathing dysfunction are poorlyunderstood. Breathing activities in the Mecp2−/y KOmouse, an animal model for RTT, during postnataldevelopment were analysed recently in detail by Viemariet al. (2005). They demonstrated plethysmographicallythat respiratory instabilities started to become obviousafter 4 weeks of age and progressed until 6 weeks of age.In the present study, we investigated the respiratory motorpattern by recording PNA and cVNA of adult KO and WTmice at postnatal day 40 ± 2 using the WHBP.

Technical considerations

The in situ WHBP of mice has been shown to reflectthe experimental condition of decerebrated in vivopreparations (Paton, 1996). In our study, we reportrelatively high PNA burst frequencies of mice preparationscompared to rats. Even in neonatal rat preparations, werarely observed such high and stable breathing frequencies(Dutschmann et al. 2000; Dutschmann & Paton, 2002).Nevertheless, ∼100 PNA bursts min−1 reflect about 50%of the breathing frequencies recorded in awake mice(∼200 breaths min−1). A similar ratio was seen for the

juvenile rat preparations (∼30 PNA bursts min−1 in situversus ∼60 breaths min−1 in vivo).

In the decerebrated WHBP, anaesthesia is not requiredand therefore the neuronal activity of the respiratorynetwork is not affected by anaesthetics. This was ofimportance for the present study, because breathingdisorders in RTT are predominantly associated with awakestates (Southall et al. 1988; Marcus et al. 1994) andapplication of anaesthesia might influence the breathingpattern of Mecp2−/y KO mice as shown by Viemari et al.(2005). Administration of low doses of pentobarbitonestabilized the respiratory pattern in KO mice (Viemariet al. 2005). Although the vigilance state of the WHBPis still undefined, the recorded respiratory motor patternsare most probably comparable to awake states because thebreathing arrhythmias recorded in the WHBP were similarto those revealed by plethysmographic measurements ofawake Mecp2−/y KO mice (Viemari et al. 2005). Thus,the WHBP is a valid model to study brainstem-linkeddisorders in Mecp2−/y KO mice.

Breathing arrhythmias correlate with impairedcontrol of postinspiration

The analysis of the respiratory motor pattern ofMecp2−/y KO preparations revealed an impaired controlof the postinspiratory activity within the respiratorycycle (also termed early expiration). The duration andamplitude of the postinspiratory vagal discharge in KOmice spontaneously fluctuated and caused unpredictablevariations in the length of the expiratory interval leading to

Figure 4. Bar diagrams illustrating the frequency (A) andduration (B) of spontaneous central apnoeasAll apnoeas are accompanied by tonic vagal postinspiratory dischargethat might indicate active laryngeal closure during the recordedbreathing pause. ∗P < 0.05; ∗∗∗P < 0.001.

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Figure 5. Apnoeas evoked by glutamate microinjections into the pontine Kolliker-Fuse nucleus (KF) inwild-type (WT) and knockout (KO) preparationsA and B, show recordings of integrated phrenic (

∫PNA) and central vagal nerve activities (

∫cVNA) to illustrate

glutamate-evoked apnoeas in representative WT and KO preparations. The glutamate-evoked apnoeas weresignificantly prolonged in KO preparations (compare grey shaded areas). In the preparations demonstrated in thisfigure, the duration of the glutamate-evoked apnoea was 5.7 s in the WT and 13.0 s in the KO preparation. Theglutamate-evoked apnoea was always accompanied by a tonic postinspiratory vagal discharge that was significantlyenhanced in KO compared to WT preparations. The anatomical location of the glutamate injection site was almostidentical. C and D, illustrate the mean apnoea duration evoked by glutamate microinjection into the KF (C) andmean accompanied integrated postinspiratory vagal discharge (D) in nine WT and 10 KO preparations. ∗P < 0.05;∗∗P < 0.01.

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breathing arrhythmias and apnoeas. By contrast, WT micedisplayed balanced phase durations of postinspirationand late expiration in order to generate a physiologicaltiming of the respiratory cycle. Postinspiratory motoroutputs specifically target laryngeal adductors in orderto control upper airway patency during the breathingcycle (Dutschmann & Paton, 2002), reflex adaptations(Shiba et al. 1999) or other behavioural challengessuch as vocalization (Farley et al. 1992; Shiba et al.

Figure 6. Semi-schematic line drawings of coronal sections through the left dorsolateral ponsDrawings, from rostral (left) to caudal (right) adapted from Paxinos & Franklin (2001), illustrate the locationof glutamate injection sites into the parabrachial complex in wild-type (WT; n = 13, A) and knockout mice(KO; n = 12, B). The drawings show glutamate-evoked apnoeas accompanied by postinspiratory tonic vagaldischarge (�), glutamate-evoked bradypnoea that was superposed to continuous enhanced vagal discharge ( )and a glutamate-evoked tachypnoea ( �). DLL, dorsal nucleus of the lateral lemniscus; Fl, flocculus; IC, inferiorcolliculus; KF, Kolliker-Fuse nucleus; LC, locus coeruleus; ll, lateral lemniscus; LPB, lateral parabrachial nucleus;LSO, lateral superior olive; Mo5, motor trigeminal nucleus; MPB, medial parabrachial nucleus; PAG, periaqueductalgrey; PFl, paraflocculus; PnC, pontine reticular nucleus, caudal part; PnO, pontine reticular nucleus, oral part; PPTg,pedunculopontine tegmental nucleus; Pr5, principal sensory trigeminal nucleus; scp, superior cerebellar peduncle;SPO, superior paraolivary nucleus; Su5, supratrigeminal nucleus.

1999; Jurgens, 2002). Thus, the impaired control ofpostinspiratory activity in KO mice is in accordance withupper airway-related phenotypes of RTT such as apnoeaswith laryngeal closure, loss of speech (Budden et al.1990) and weak coordination of breathing and swallowing(Morton et al. 1997; Isaacs et al. 2003).

Postinspiratory activity is essentially determinedby inhibitory synaptic inputs from defined neuronalpopulations (e.g. decrementing inspiratory or augmenting

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expiratory neurones; Ezure, 1990; Richter et al. 1992)within the mammalian respiratory network. However,strength and duration of the postinspiratory activity areregulated by two excitatory mechanisms. One is the gatingof postinspiratory network activity, via descending controlof the excitability of medullary postinspiratory neurones,arising from the pontine KF (Dutschmann & Herbert,2006). The other is the activation of postinspiratoryneurones by peripheral inputs from pulmonary stretchreceptors during the late stages of lung inflation (Hayashiet al. 1996; Ezure, 2004). Results from the presentstudy suggest severe impairments of both mechanismsin Mecp2−/y KO mice. Glutamate injections into the KFevoked transient postinspiratory apnoeas in WT andKO mice but the glutamate-evoked responses were farmore pronounced in KO preparations. As the perfusedpreparation is lacking any feedback from pulmonarystretch receptors, the generation of disturbed respiratorymotor pattern of KO mice might be predominantly causedby a hyperexcitability of KF neurones. Under intact invivo conditions, such an impairment of the KF might becompensated by afferent feedback from pulmonary stretchreceptors. However, central vagal nerve stimulation inorder to mimic the HBR of the pulmonary stretch receptorsalso revealed exaggerated apnoeic responses in KO mice.Thus, the severe respiratory phenotype of Mecp2−/y

KO mice might be caused by an imbalance of sensorypathways and network intrinsic circuits controlling thepostinspiratory activity.

Potential synaptic imbalances underlying thebreathing disorders in RTT

Only limited information about pathological changes inbrainstem transmitter systems or synaptic functions inRTT are available. The data suggest altered expression

Figure 7. Fictive Hering-Breuer reflex (HBR) and desensitization of the sensory reflex apnoea in responseto repetitive vagal stimulationA and B, illustrate phrenic nerve activity (PNA) recordings of a representative wild-type (WT) and knockout (KO)preparation before, during and after repetitive electrical stimulation of the central vagal nerve (15 stimulationtrials every 60 s, 10 s duration of stimulation). The upper traces show the first vagal stimulation trial, the lowertraces the 15th stimulation trial. A clear desensitization of the HBR was observed in all WT preparations. In thedisplayed example, the duration of the evoked apnoea decreased from 10.19 to 4.47 s during the course of 15stimulation trials (A). By contrast, KO preparations showed a significant prolonged sensory reflex apnoea after thefirst stimulation trial and desensitization of the HBR was never observed. The duration of the sensory evoked apnoeain the demonstrated KO preparation (B) remained constant between the first and last stimulation trial (16.39 versus16.12 s). C, illustrates the significant differences in the duration of the evoked apnoea in response to the initialstimulation trial between KO and WT mice. ∗∗∗P < 0.001. D, demonstrates the duration of evoked apnoeas duringthe 10 s of repetitive vagal stimulation. KO preparations never showed any sign of desensitization and had constantapnoea duration of ∼10 s during all trials of 10 s stimulation. By contrast, the regression of apnoea duration in WTpreparations demonstrated a clear stimulus-associated reflex desensitization. This desensitization occurred rapidlyduring the first five stimulation trials. Apnoea duration thereafter remained constant. Note that in contrast to theother data, the 99.7% confidence interval is shown. This confidence interval automatically corrects for 15 multipletrials according to Bonferroni and demonstrates significance for the comparison between WT and KO mice if theconfidence intervals are not overlapping.

Figure 8. Simplified model for disturbed synaptic activity in theponto-medullary respiratory network in RTTPopulations of different respiratory neurons are shown (white circles).Highlighted is the potentially disturbed gating of postinspiratoryactivity via reciprocal excitatory interactions of pontine and medullarysubsets of postinspiratory neurones. The disturbed gating might relatein particular to upper airway-related disorders of RTT such as apnoeaswith laryngeal closure, loss of speech and weak coordination ofbreathing and swallowing (for details see Discussion). Lines witharrows or black circles show excitatory (glutamate/NMDA) or inhibitory(GABA, glycine) synaptic connections, respectively. Glu, glutamate;Gly, glycine; I, inspiration; PI, postinspiration; E2, late expiration; NTS,nucleus of the solitary tract; PSR, pulmonary stretch reflex; HBR,Hering-Breuer reflex; , overexcitation/disinhibition.

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874 G. M. Stettner and others J Physiol 579.3

of neuropeptides (e.g. substance P or met-enkephalin;Saito et al. 2001) or pathological changes in serotonergicand nor-adrendergic transmitter systems (Lekman et al.1989; Zoghbi et al. 1989; Paterson et al. 2005; Ideet al. 2005; Viemari et al. 2005). These neuropeptidesand neurotransmitters have important functions ingeneration and modulation of the respiratory rhythm.Altered expression profiles may therefore contribute to thebreathing phenotype in RTT.

Results from the present study imply a hyperactivityof postinspiratory neurones which, according to currenthypotheses, are not directly involved in rhythm generation(Onimaru & Homma, 2003; Duffin, 2004; Feldman& Del Negro, 2006). Postinspiratory activity is largelydetermined by glutamatergic peripheral synaptic inputsand ponto-medullary interactions in which NMDAreceptors play a key role (see model Fig. 8). Interestingly,an MeCP2 deficiency-related alteration in the expressionof specific NMDA receptor subunits in the hippocampuswas demonstrated recently (Asaka et al. 2006). Similarchanges in the KF could contribute to the observed excessof excitation because the KF and parabrachial nucleihave been shown to densely express NMDA receptors(Monaghan & Cotman, 1985; Guthmann & Herbert,1999). Blockade of pontine NMDA receptors causes asevere disruption of the motor pattern affecting thetiming of the inspiratory (prolonged) and expiratory(shortened) phases (for review see St-John & Paton, 2004;Rybak et al. 2004). By contrast, a hyperexcitability inthe pontine gating mechanisms of the postinspiratoryactivity due to up-regulation or alteration in thesubunit composition of NMDA receptors within the KFprovokes the opposite effect: premature termination ofinspiration and a prolonged and highly variable expiratoryduration, as seen in Mecp2−/y KO mice. Furthermore,changes in NMDA receptor expression in the primaryrelay centres for pulmonary stretch receptors within thenucleus of the solitary tract or in secondary relays withinthe KF (Miyazaki et al. 1999; Ezure, 2004; Song & Poon,2004) potentially contribute to the pathological increasedsensitivity of the HBR observed in KO mice.

Other explanations for excessive excitability ofpostinspiratory activity are possible from the finding thatGABA receptor subunit-related genes are down-regulatedin Mecp2−/y KO mice (Samaco et al. 2005). The reciprocalGABAergic and glycinergic synaptic inhibition betweensubsets of respiratory neurones required for oscillationof the network during rhythm and pattern generation(for review see Richter, 2003) might be impaired (Fig. 8).Consequently a decrease in GABAergic inhibition ofmedullary or pontine postinspiratory neurones would leadto disinhibition and therefore hyperexcitability.

Overall, the most likely explanation for RTT-associatedbreathing disorders is an inhibitory and excitatoryimbalance of ponto-medullary synaptic interactions that

are required for the generation of a normal respiratorymotor pattern. Such inhibitory and excitatory imbalanceswere found in pyramidal neurones in Mecp2−/y KO mice,although in the cortical regions the balance was shifted toenhanced inhibition (Dani et al. 2005). In the respiratorynetwork, however, the imbalance is instead altered towardsexcitation.

Disturbed maturation of the respiratory network

In patients with RTT, breathing abnormalities becomeobvious after an initially near-normal psychomotordevelopment and a regression period, which occursbetween 1 and 3 years of age, suggesting progressiveimpairment of synaptic functions during the postnataldevelopment of the respiratory network. In particular,the natural maturation of the respiratory network isaccompanied by significant changes in the expressionprofiles of NMDA, GABA and glycine receptors. It isapparent that the expression of NMDA receptors decreases,while the expression of inhibitory receptors increasesduring postnatal development (for review see Wong-Riley& Liu, 2005). Regarding RTT, there is growing evidencethat the transcription repressor MeCP2 is connected todevelopmental changes in the expression of NMDA andGABA receptor subunits (Samaco et al. 2005; Young et al.2005). Thus, MECP2 mutations might cause the abovesuggested overexcitability of postinspiratory neurones.

A pathological regulation of NMDA receptors alsomight contribute to impaired synaptic plasticity inMecp2−/y KO mice as demonstrated in conjunction withlong-term potentiation in the hippocampus (Asaka et al.2006; Moretti et al. 2006). Our results indicate a lack ofdesensitization of the HBR in KO mice and also suggestdisturbed plasticity within the respiratory network.Certain forms of short-term synaptic plasticity withinthe respiratory network undergo substantial maturation(Dutschmann et al. 2004). A disturbed maturation of thesenetwork circuits in RTT might therefore particularly affectplasticity dependent adaptive behaviour of the respiratorynetwork.

Conclusions

The main finding of our study is a dysfunction of thecentral and vagal control of the postinspiratory activityin Mecp2−/y KO mice. The results are of clinical interestbecause most of the respiratory abnormalities in patientswith RTT including repetitive apnoeas, breath-holdingspells, Valsalva’s manoeuvres and loss of speech canpotentially be explained by similar pathophysiologicalmechanisms, which are the main basis for devising effectivepharmacological therapies.

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Acknowledgements

G. Stettner is supported by a postdoctoral fellowship award

of the Rett Syndrome Research Foundation (RSRF, Cincinnati,

OH, USA). The study was supported by a grant from the

Deutsche Forschungsgemeinschaft (DFG; HU 941/2–1) and the

DFG Research Center for Molecular Physiology of the Brain

(CMPB, Gottingen, Germany). The authors would like to thank

A. Bischoff for excellent technical assistance, E. Munk for animal

breeding and husbandry, and K. Kohler (Department of Genetic

Epidemiology, Georg August University Gottingen) for statistical

advice.

Disclosure

The authors have reported no conflicts of interest.

C© 2007 The Authors. Journal compilation C© 2007 The Physiological Society