Breathing dysfunctions associated with impaired control of postinspiratory activity in Mecp2−/y knockout mice
Abstract
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 Kölliker-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.
———
Rett syndrome (RTT) is an X-linked neurodevelopmental disorder associated with breathing abnormalities and severe mental retardation in females (
;
). Mutations and gene deletions in the MECP2 gene located on Xq28 have been identified as primary causes of the disease (
). The MECP2 gene encodes the methyl-CpG binding protein 2 (MeCP2), which acts as a transcriptional repressor. The target genes regulated by MeCP2 are still incompletely known and subject to intensive research (for review see
).
The clinical course of RTT is characterized by different stages (
Hagberg & Witt Engerström, 1986
). Towards the end of the regression period, which occurs between 1 and 3 years of age, most patients develop state-dependent breathing abnormalities. These abnormalities are included in the clinical diagnostic criteria for RTT (
). Respiratory disturbances during wakefulness comprise alternating periods of hyperventilation and apneustic, breath-holding frequently terminated by Valsalva's manoeuvres and forced and deep breathing, as well as apnoeic breathing (e.g.
;
;
;
). Importantly, these respiratory arrhythmias are seen as a main cause of sudden and unexpected deaths in RTT patients (
). By contrast, breathing during sleep is apparently stable (
;
).
In spite of the clinical impact of the respiratory dysfunction in RTT, little is known about the underlying pathomechanisms. Now, the availability of the Mecp2−/y knockout (KO) mouse (
;
;
), an animal model for human RTT, allows analytic studies of respiratory dysfunction.
showed recently that Mecp2−/y KO mice have an RTT-like respiratory disorder. The primary cause of the breathing abnormalities in mice is as unclear as in patients. There is some support for the hypothesis of cortical dysfunction and thus behaviourally associated respiratory dysfunction (
;
); however, others have reported brainstem immaturity (
;
) or disturbance of neuromodulatory regulation of synaptic function within the ponto-medullary respiratory networks (
).
In the present study we investigated the respiratory motor pattern of Mecp2−/y KO mice and control C57BL/6J wild-type (WT) mice using the decerebrated and arterially perfused working heart–brainstem preparation (WHBP;
). Because of the decerebration, potential influences of higher brain regions on the in situ respiratory pattern generators of the ponto-medullary brainstem are excluded. RTT patients also have upper airway-related clinical problems, such as loss of speech and impaired swallowing. Thus we were also interested in the pontine circuits (e.g. Kölliker-Fuse nucleus (KF), see
) and sensory inputs (Hering-Breuer reflex (HBR)) that control postinspiratory mediated activation of laryngeal adductors. Our results demonstrate a significant impairment of the pontine and sensory control of the postinspiratory motor activities in Mecp2−/y KO mice preparations.
Methods
The experimental procedures were performed in accordance with European community and National Institutes of Health guidelines for the care and use of laboratory animals. The ethical committee of the Georg August University Göttingen approved the study.
The developmental course of the breathing dysfunction in Mecp2−/y KO mice has previously been described by
. Within 6 weeks of age, the KO mice develop a severe RTT-like breathing phenotype. Our study has not focused on the development but rather the range of respiratory dysfunctions in KO mice. Thus, we decided to work on adult KO and WT mice at a postnatal age of 40 ± 2 days.
Animal breeding and genotyping
Experiments were performed using the mouse model for RTT, strain B6.129P2(C)-Mecp2tm1–1Bird (
). The mice were obtained from The Jackson Laboratory (Bar Harbour, ME, USA) and maintained on a C57BL/6J background. Hemizygous mutant Mecp2−/y males were generated by crossing heterozygous Mecp2+/− females with C57BL/6J WT males. All mice were routinely genotyped in accordance with The Jackson Laboratory genotyping protocols (
http://jaxmice.jax.org/strain/003890.html
). For these purposes genomic DNA was isolated from mice tails using the DNeasy Tissue Kit (Qiagen, Hilden, Germany) according to manufacturer's protocol.
All experiments were performed in hemizygous Mecp2−/y KO and C57BL/6J WT male mice. We exclusively used Mecp2−/y KO males because heterozygous Mecp2+/− female mice have an unpredictable and heterogeneous phenotype due to the X-inactivation profile of the X-linked MECP2 gene.
WHBP
The experiments were performed using the arterially perfused WHBP. Mice were anaesthetized deeply in a saturated atmosphere of isoflurane (1-chloro-2,2,2-trifluoroethyl-difluoromethylether; Abbott, Wiesbaden, Germany). Once the animal failed to respond to noxious pinch to the tail or a hind paw, it was transected below the diaphragm, transferred into ice-cooled (5°C) artificial cerebrospinal fluid (aCSF) gassed with carbogen (95% O2– 5% CO2), decerebrated at the precollicular level and cerebellectomised. The lungs were removed. The left phrenic nerve (PN) was separated and cut at the level of the diaphragm. The descending aorta was isolated from the ventral surface of the spinal column. These initial procedures took approximately 10 min. The preparation was then transferred to a recording chamber. The descending aorta was cannulated and perfused using a peristaltic pump (Watson-Marlow, Wilmington, MA, USA) with carbogen-gassed aCSF at 31°C containing Ficoll (1.25%, Sigma-Aldrich, Steinheim, Germany) to maintain colloidal 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. The osmolarity of the perfusate was 300 ± 10 mosmol l−1 and the pH was adjusted to 7.35 ± 0.05 by gassing with carbogen. The perfusate was filtered and passed through bubble traps to remove gas bubbles. The perfusate leaking from the preparation was collected and recirculated after reoxygenation. Cardiac activity returned within seconds and rhythmic contractions of respiratory muscles within a few minutes after onset of reperfusion. Respiratory related movements were abolished by vecuronium bromide (0.3 μg ml−1, Inresa, Freiburg i. Br., Germany).
After paralysis, phrenic nerve activity (PNA) was primarily used to fine-tune the preparations by adjusting the flow rate (10–22 ml min−1) and perfusion pressure (40–70 mmHg). For measurement of aortic perfusion pressure, a double-lumen catheter was used and connected to a pressure transducer. During the tuning, the flow rates were individually adjusted to fulfil the following criteria: re-appearance of a clearly identifiable three-phase respiratory pattern, rhythmic respiratory motor discharges of at least 60 bursts min−1 and maintenance of the respiratory pattern (although disturbed in KO mice) for at least 30 min before starting any experiments or data analysis. In addition during the tuning phase, the right central vagus nerve (cVN) was dissected and prepared for recording.
Nerve and cardiac recordings
Respiratory motor nerve activities were recorded simultaneously from the central ends of the phrenic and vagal nerves in all preparations via suction electrodes connected to differential amplifiers (Neurolog 100). Nerve activity was amplified and filtered (8 Hz to 3 kHz; Neurolog modules 104 and 125). All data were digitized using a MacLab 8s interface and stored on a computer using Chart software (version 5.2, ADInstruments, Australia). Respiratory motor nerve activity was integrated (time constant, 100 ms). The electrocardiogram was recorded simultaneously with the PNA.
Data analysis
Using the PNA and central vagal nerve activity (cVNA) recordings we analysed the duration of the following respiratory parameters: total respiratory cycle length (Ttot), inspiration (Ti), postinspiration (Tpi) and late expiration (Te2). In contrast to in vivo investigations in the cat, PNA recording does not display a clearly identifiable postinspiratory activity in mice. Therefore we classified the entire expiratory interval as Te (including Tpi and Te2). Tpi and Te2 were identified and measured from the cVNA. The inspiratory cVNA coincided with the PNA. The duration of the expiratory cVNA was defined as Tpi, and the phase with absent cVNA in late expiration as Te2. Breathing frequency was indicated by the PNA (bursts min−1). In addition to the duration we analysed the variance of the respiratory parameters. These parameters were calculated from 60 consecutive breathing cycles at a representative control activity. Furthermore, we measured the frequency and duration of spontaneous apnoeas over a period of at least 5 min. We defined an apnoea as the absence of rhythmic PNA for a period of at least two respiratory cycles based on the mean Ttot calculated from 60 consecutive breathing cycles at a representative control activity. Because we focused on the respiratory dysfunction in Mecp2−/y KO mice in the present study, we did not analyse 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 pontine KF were performed with a micromanipulator-driven multi-barrelled micropipette (tip diameter, 30–40 μm). The micropipette was placed just caudally to the left inferior colliculus and 0.6–0.7 mm medial from the lateral margin of the pons. The depth of the microinjection was 1.4–1.6 mm. The injected volumes were measured by observing the descending drug meniscus in the micropipette through a binocular microscope fitted with a calibrated eyepiece graticule. After identification of the effective injection site (e.g. by transient apnoea or tachypnoea following glutamate injection), pontamine sky blue was injected to mark the area of microinjection. The duration of the glutamate-evoked apnoea and the integral discharge of the postinspiratory cVNA during the glutamate-evoked apnoea were analysed. The end of the glutamate-evoked apnoea was defined by the reappearance of the first PNA burst. At the end of each experiment, the brainstem was removed and fixed in 4% paraformaldehyde (Merck, Darmstadt, Germany) containing 20% sucrose (Merck). For anatomical verification of the injection sites we cut series of 50 μm thick coronal sections through the pons with a freezing microtome (Reichert and Jung, Wetzlar, Germany). Afterwards, the sections were stained with neutral red solution (Sigma-Aldrich). The locations of microinjections were documented on semi-schematic drawings of coronal sections through the dorsolateral pons adapted from
.
Electrical stimulation of vagal afferents
To mimic the HBR the central branch of the left vagal nerve was electrically stimulated through a suction electrode. The indifferent electrode was placed into the cervical tissue near to the vagal nerve. We analysed the duration of the sensory evoked reflex apnoea as seen in the PNA and the desensitization of the postinspiratory reflex apnoea in response to repetitive vagal stimulation (15 trials every 60 s, 10 s duration of each stimulation trial, 100 μs pulse width, 20 Hz frequency, 0.4–4 mV).
Statistical analysis
Analysis was performed with the Statistica 7.1 software package (StatSoft Inc., Tulsa, OK, USA). All results are presented as means ±s.e.m. Respiratory parameters of WT and KO preparations were compared using unpaired Student's t test in the case of normally distributed data (phase durations, frequency and duration of spontaneous apnoeas, duration of sensory and glutamate-evoked apnoea) and using the Mann–Whitney U test in the case of non-normally distributed data (integrated postinspiratory vagal discharge during glutamate-evoked apnoea). To indicate the scale of phase durations, the variance of phase durations measured from 60 consecutive breathing cycles of every preparation (see data analysis) was calculated. The variances of the phase durations in WT and KO mice were compared using the Mann–Whitney U test because of non-normal distribution of the data. Correlations between two phase durations measured from 60 consecutive breathing cycles of every individual preparation (see data analysis) were investigated using Pearson's correlation coefficient. All data sets were tested for normal distribution prior to the correlation analysis. For comparison of the correlation coefficients between the groups of WT and KO mice, the Mann–Whitney U test was used because of non-normal data distribution. P < 0.05 was considered significant.
Results
In the present study we analysed and compared the respiratory phenotype of Mecp2−/y KO and their control C57BL/6J WT male mice using the arterially perfused WHBP. For our investigations we used 28 KO and 28 WT male mice at postnatal day 40 ± 2. Simultaneous recordings of PNA and cVNA were performed in all preparations.
Characterization of the respiratory motor pattern
Both WT (n = 28) and KO preparations (n = 28) generated a respiratory activity characterized by an incrementing inspiratory PNA and a biphasic inspiratory and postinspiratory vagal discharge pattern (
).
Figure 1.
Recording of the phrenic (PNA) and central vagal nerve activities (cVNA) of representative wild-type (WT) and knockout (KO) preparations The figure shows traces of 20 s duration of a WT and KO preparation (A and C) as well as sections with enlarged time scale of the same preparations to demonstrate differences in the respiratory phase durations (B and D). A and B, display the eupnoeic breathing pattern of a WT mouse with an incrementing discharge of the phrenic nerve during inspiration and biphasic inspiratory and postinspiratory discharge of the vagal nerve. There is no discharge in both recorded nerves during the late expiratory phase. C and D, illustrate characteristic features of respiratory abnormalities in a KO preparation. The rhythm is irregular due to a mixture of fast and slow breathing activity and repetitive apnoeas (*). Please note the variability of vagal postinspiratory discharge in terms of duration and amplitude (+). D, shows a section with enlarged time scale of stable breathing of the same KO preparation as in C. PNA burst frequency is identical to the WT preparation (see B), but inspiratory duration (Ti) is slightly shortened and postinspiratory duration (Tpi) markedly prolonged.
WT preparations displayed a fast and regular respiratory rhythm with a PNA burst frequency of 99.1 ± 5.5 min−1 (
and
). Analysis of the phase durations as determined from PNA and cVNA revealed the following values: Ti, 0.20 ± 0.01 s; Tpi, 0.24 ± 0.02 s; Te2, 0.21 ± 0.01 s; Te, 0.45 ± 0.03 s; Ttot, 0.65 ± 0.03 s (
). Overall, there was a regular three-phase discharge and the phase relations suggested an eupnoeic respiratory motor pattern.
Figure 3.
Sequential plot of duration of postinspiration (Tpi) and total respiratory cycle length (Ttot) 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 Ttot values are regular in the WT mouse but scattered in the KO mouse. Prolonged Tpi values correlate with prolonged Ttot values.
Figure 2.
Bar diagrams illustrating mean phase durations ±s.e.m. (A) and mean variance of phase durations ±s.e.m. (B) in 28 wild-type (WT) and 28 knockout (KO) preparations Please note the logarithmic scale of the y-axis in B. Ti, duration of inspiration; Tpi, duration of postinspiration; Te2, duration of late expiration; Te, duration of the entire expiratory interval; Ttot, total respiratory cycle length; n.s., not significant; *P < 0.05; ***P < 0.001.
In KO preparations the mean durations of the inspiratory, postinspiratory and late expiratory phases showed highly significant differences while mean Te (0.50 ± 0.03 s), Ttot (0.67 ± 0.04 s) and burst frequency of PNA (97.4 ± 5.5 min−1) did not differ significantly from those in WT preparations. In particular, the mean Tpi was markedly prolonged (0.41 ± 0.03 s, P < 0.001) whereas the mean inspiratory (0.17 ± 0.01 s, P < 0.05) and late expiratory (0.08 ± 0.01 s, P < 0.001) phase durations in KO preparations were shortened (
).
All KO preparations showed pronounced respiratory arrhythmia (
and
). The arrhythmia was characterized by a highly significant increase in variance of Ttot, Tpi and Ti (each P < 0.001) in comparison to WT preparations (
). During the arrhythmia individual prolonged Tpi values correlated with prolonged Ttot values (
). However, Te2 did not affect the respiratory arrhythmia in KO preparations because there was no difference in the variance of Te2 when compared to WT controls.
In KO and WT preparations, a relationship between mean Tpi and Ttot values was observed. However, the mean correlation coefficient between Tpi and Ttot measured from 60 consecutive breathing cycles of each preparation was significantly higher in KO than in WT mice (r = 0.97 ± 0.01 versus r = 0.66 ± 0.04, P < 0.001). Almost identical correlation coefficients and a similar level of significance were calculated for correlations between Tpi and Te. There was no correlation between Te2 and Ttot values in KO preparations, whereas WT preparations showed a marked correlation (r = 0.22 ± 0.03 versus r = 0.65 ± 0.03, P < 0.001). These results indicate that Tpi mainly determines Te and therefore Ttot in KO mice.
All WT preparations displayed unprovoked transient central apnoeas accompanied by tonic vagal postinspiratory discharge. These apnoeas occurred at a frequency of 0.85 ± 0.10 apnoeas min−1. By contrast, KO preparations showed such repetitive apnoeas at a significantly higher frequency (2.45 ± 0.35 apnoeas min−1, P < 0.001,
). The mean duration of spontaneous central apnoeas in WT preparations was 2.73 ± 0.18 s, accounting for a 4.24 ± 0.24-fold increase in the mean Ttot. In KO preparations, the spontaneous apnoeas lasted for 2.13 ± 0.14 s, accounting for a 3.22 ± 0.15-fold increase in the mean Ttot (P < 0.05,
).
Figure 4.
Bar diagrams illustrating the frequency (A) and duration (B) of spontaneous central apnoeas All apnoeas are accompanied by tonic vagal postinspiratory discharge that might indicate active laryngeal closure during the recorded breathing pause. *P < 0.05; ***P < 0.001.
Pontine glutamate microinjections
We injected glutamate (10 mm, 40 nl) into the area of the left KF in 13 WT and 12 KO preparations. All injection sites were marked with pontamine sky blue after glutamate injection using multi-barrelled micropipettes. The histologically verified locations of microinjections were documented on semi-schematic drawings of coronal sections through the dorsolateral pons (
).
Figure 6.
Semi-schematic line drawings of coronal sections through the left dorsolateral pons Drawings, from rostral (left) to caudal (right) adapted from
, illustrate the location of 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 vagal discharge (⋆), 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, inferior colliculus; KF, Kölliker-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, periaqueductal grey; 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.
All glutamate injections that triggered a transient postinspiratory apnoea were clustered in the intermediate part of KF (KO, n = 10; WT, n = 9; stars in
). The evoked apnoeas following glutamate injections of comparable volumes into almost identical areas of the intermediate KF (see
) were significantly longer in KO when compared to WT preparations (17.37 ± 3.32 s versus 4.60 ± 0.63 s, P < 0.01,
). In all preparations, the glutamate-evoked respiratory arrest was accompanied by a tonic postinspiratory vagal discharge. This tonic vagal activity was significantly enhanced in KO when compared to WT preparations as indicated by the integrated vagal activity during the glutamate-evoked apnoea (47.4 ± 3.3 versus 12.5 ± 2.8 mV × 100 ms, P < 0.05,
).
Figure 5.
Apnoeas evoked by glutamate microinjections into the pontine Kölliker-Fuse nucleus (KF) in wild-type (WT) and knockout (KO) preparations A 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 were significantly prolonged in KO preparations (compare grey shaded areas). In the preparations demonstrated in this figure, the duration of the glutamate-evoked apnoea was 5.7 s in the WT and 13.0 s in the KO preparation. The glutamate-evoked apnoea was always accompanied by a tonic postinspiratory vagal discharge that was significantly enhanced in KO compared to WT preparations. The anatomical location of the glutamate injection site was almost identical. C and D, illustrate the mean apnoea duration evoked by glutamate microinjection into the KF (C) and mean accompanied integrated postinspiratory vagal discharge (D) in nine WT and 10 KO preparations. *P < 0.05; **P < 0.01.
Injections that did not produce postinspiratory apnoeas were mainly located in the margins of the KF area and resulted in bradypnoea with enhanced postinspiratory activity (diamonds in
). In a single preparation, glutamate injection into the KF produced a tachypnoea without additional postinspiratory modulation (open circle in
).
Fictive HBR and desensitization in response to repetitive vagal stimulation
We evoked a fictive HBR by electrical stimulation of the central branch of the vagal nerve in nine WT and nine KO mice. When compared to WT controls, the sensory evoked apnoeas in KO preparations were all prolonged and lasted for several seconds after the stimulus off-switch (
). The first stimulation trial revealed a mean duration of reflex apnoea including the 10 s vagal stimulation of 19.86 ± 2.43 s in KO preparations while the apnoea was significantly shorter in WT preparations (10.43 ± 0.16 s, P < 0.001,
). All WT preparations showed a clear desensitization of the HBR in response to repetitive vagal stimulation (15 consecutive stimulation trials every 60 s, 10 s duration of stimulation) with a shortening of the mean evoked reflex apnoea from 10.43 ± 0.16 to 3.92 ± 0.50 s (
). By contrast, KO preparations did not show any sign of desensitization (
).
Figure 7.
Fictive Hering-Breuer reflex (HBR) and desensitization of the sensory reflex apnoea in response to repetitive vagal stimulation A 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 stimulation trials every 60 s, 10 s duration of stimulation). The upper traces show the first vagal stimulation trial, the lower traces the 15th stimulation trial. A clear desensitization of the HBR was observed in all WT preparations. In the displayed example, the duration of the evoked apnoea decreased from 10.19 to 4.47 s during the course of 15 stimulation trials (A). By contrast, KO preparations showed a significant prolonged sensory reflex apnoea after the first stimulation trial and desensitization of the HBR was never observed. The duration of the sensory evoked apnoea in the demonstrated KO preparation (B) remained constant between the first and last stimulation trial (16.39 versus 16.12 s). C, illustrates the significant differences in the duration of the evoked apnoea in response to the initial stimulation trial between KO and WT mice. ***P < 0.001. D, demonstrates the duration of evoked apnoeas during the 10 s of repetitive vagal stimulation. KO preparations never showed any sign of desensitization and had constant apnoea duration of ∼10 s during all trials of 10 s stimulation. By contrast, the regression of apnoea duration in WT preparations demonstrated a clear stimulus-associated reflex desensitization. This desensitization occurred rapidly during the first five stimulation trials. Apnoea duration thereafter remained constant. Note that in contrast to the other data, the 99.7% confidence interval is shown. This confidence interval automatically corrects for 15 multiple trials according to Bonferroni and demonstrates significance for the comparison between WT and KO mice if the confidence intervals are not overlapping.
Discussion
Most patients suffering from the inborn neurodevelopmental disorder RTT also develop severe breathing arrhythmias. The pathological mechanisms of RTT-associated breathing dysfunction are poorly understood. Breathing activities in the Mecp2−/y KO mouse, an animal model for RTT, during postnatal development were analysed recently in detail by
. They demonstrated plethysmographically that respiratory instabilities started to become obvious after 4 weeks of age and progressed until 6 weeks of age. In the present study, we investigated the respiratory motor pattern by recording PNA and cVNA of adult KO and WT mice at postnatal day 40 ± 2 using the WHBP.
Technical considerations
The in situ WHBP of mice has been shown to reflect the experimental condition of decerebrated in vivo preparations (
). In our study, we report relatively high PNA burst frequencies of mice preparations compared to rats. Even in neonatal rat preparations, we rarely observed such high and stable breathing frequencies (
;
). 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−1in situ versus∼60 breaths min−1in vivo).
In the decerebrated WHBP, anaesthesia is not required and therefore the neuronal activity of the respiratory network is not affected by anaesthetics. This was of importance for the present study, because breathing disorders in RTT are predominantly associated with awake states (
;
) and application of anaesthesia might influence the breathing pattern of Mecp2−/y KO mice as shown by
. Administration of low doses of pentobarbitone stabilized the respiratory pattern in KO mice (
). Although the vigilance state of the WHBP is still undefined, the recorded respiratory motor patterns are most probably comparable to awake states because the breathing arrhythmias recorded in the WHBP were similar to those revealed by plethysmographic measurements of awake Mecp2−/y KO mice (
). Thus, the WHBP is a valid model to study brainstem-linked disorders in Mecp2−/y KO mice.
Breathing arrhythmias correlate with impaired control of postinspiration
The analysis of the respiratory motor pattern of Mecp2−/y KO preparations revealed an impaired control of the postinspiratory activity within the respiratory cycle (also termed early expiration). The duration and amplitude of the postinspiratory vagal discharge in KO mice spontaneously fluctuated and caused unpredictable variations in the length of the expiratory interval leading to breathing arrhythmias and apnoeas. By contrast, WT mice displayed balanced phase durations of postinspiration and late expiration in order to generate a physiological timing of the respiratory cycle. Postinspiratory motor outputs specifically target laryngeal adductors in order to control upper airway patency during the breathing cycle (
), reflex adaptations (
) or other behavioural challenges such as vocalization (
;
;
). Thus, the impaired control of postinspiratory activity in KO mice is in accordance with upper airway-related phenotypes of RTT such as apnoeas with laryngeal closure, loss of speech (
) and weak coordination of breathing and swallowing (
;
).
Postinspiratory activity is essentially determined by inhibitory synaptic inputs from defined neuronal populations (e.g. decrementing inspiratory or augmenting expiratory neurones;
;
) within the mammalian respiratory network. However, strength and duration of the postinspiratory activity are regulated by two excitatory mechanisms. One is the gating of postinspiratory network activity, via descending control of the excitability of medullary postinspiratory neurones, arising from the pontine KF (
). The other is the activation of postinspiratory neurones by peripheral inputs from pulmonary stretch receptors during the late stages of lung inflation (
;
). Results from the present study suggest severe impairments of both mechanisms in Mecp2−/y KO mice. Glutamate injections into the KF evoked transient postinspiratory apnoeas in WT and KO mice but the glutamate-evoked responses were far more pronounced in KO preparations. As the perfused preparation is lacking any feedback from pulmonary stretch receptors, the generation of disturbed respiratory motor pattern of KO mice might be predominantly caused by a hyperexcitability of KF neurones. Under intact in vivo conditions, such an impairment of the KF might be compensated by afferent feedback from pulmonary stretch receptors. However, central vagal nerve stimulation in order to mimic the HBR of the pulmonary stretch receptors also revealed exaggerated apnoeic responses in KO mice. Thus, the severe respiratory phenotype of Mecp2−/y KO mice might be caused by an imbalance of sensory pathways and network intrinsic circuits controlling the postinspiratory activity.
Potential synaptic imbalances underlying the breathing disorders in RTT
Only limited information about pathological changes in brainstem transmitter systems or synaptic functions in RTT are available. The data suggest altered expression of neuropeptides (e.g. substance P or met-enkephalin;
) or pathological changes in serotonergic and nor-adrendergic transmitter systems (
;
;
;
;
). These neuropeptides and neurotransmitters have important functions in generation and modulation of the respiratory rhythm. Altered expression profiles may therefore contribute to the breathing phenotype in RTT.
Results from the present study imply a hyperactivity of postinspiratory neurones which, according to current hypotheses, are not directly involved in rhythm generation (
;
;
). Postinspiratory activity is largely determined by glutamatergic peripheral synaptic inputs and ponto-medullary interactions in which NMDA receptors play a key role (see model
). Interestingly, an MeCP2 deficiency-related alteration in the expression of specific NMDA receptor subunits in the hippocampus was demonstrated recently (
). Similar changes in the KF could contribute to the observed excess of excitation because the KF and parabrachial nuclei have been shown to densely express NMDA receptors (
;
). Blockade of pontine NMDA receptors causes a severe disruption of the motor pattern affecting the timing of the inspiratory (prolonged) and expiratory (shortened) phases (for review see
;
). By contrast, a hyperexcitability in the pontine gating mechanisms of the postinspiratory activity due to up-regulation or alteration in the subunit composition of NMDA receptors within the KF provokes the opposite effect: premature termination of inspiration and a prolonged and highly variable expiratory duration, as seen in Mecp2−/y KO mice. Furthermore, changes in NMDA receptor expression in the primary relay centres for pulmonary stretch receptors within the nucleus of the solitary tract or in secondary relays within the KF (
;
;
) potentially contribute to the pathological increased sensitivity of the HBR observed in KO mice.
Figure 8.
Simplified model for disturbed synaptic activity in the ponto-medullary respiratory network in RTT Populations of different respiratory neurons are shown (white circles). Highlighted is the potentially disturbed gating of postinspiratory activity via reciprocal excitatory interactions of pontine and medullary subsets of postinspiratory neurones. The disturbed gating might relate in particular to upper airway-related disorders of RTT such as apnoeas with laryngeal closure, loss of speech and weak coordination of breathing and swallowing (for details see Discussion). Lines with arrows 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.
Other explanations for excessive excitability of postinspiratory activity are possible from the finding that GABA receptor subunit-related genes are down-regulated in Mecp2−/y KO mice (
). The reciprocal GABAergic and glycinergic synaptic inhibition between subsets of respiratory neurones required for oscillation of the network during rhythm and pattern generation (for review see
) might be impaired (
). Consequently a decrease in GABAergic inhibition of medullary or pontine postinspiratory neurones would lead to disinhibition and therefore hyperexcitability.
Overall, the most likely explanation for RTT-associated breathing disorders is an inhibitory and excitatory imbalance of ponto-medullary synaptic interactions that are required for the generation of a normal respiratory motor pattern. Such inhibitory and excitatory imbalances were found in pyramidal neurones in Mecp2−/y KO mice, although in the cortical regions the balance was shifted to enhanced inhibition (
). In the respiratory network, however, the imbalance is instead altered towards excitation.
Disturbed maturation of the respiratory network
In patients with RTT, breathing abnormalities become obvious after an initially near-normal psychomotor development and a regression period, which occurs between 1 and 3 years of age, suggesting progressive impairment of synaptic functions during the postnatal development of the respiratory network. In particular, the natural maturation of the respiratory network is accompanied by significant changes in the expression profiles of NMDA, GABA and glycine receptors. It is apparent that the expression of NMDA receptors decreases, while the expression of inhibitory receptors increases during postnatal development (for review see
). Regarding RTT, there is growing evidence that the transcription repressor MeCP2 is connected to developmental changes in the expression of NMDA and GABA receptor subunits (
;
). Thus, MECP2 mutations might cause the above suggested overexcitability of postinspiratory neurones.
A pathological regulation of NMDA receptors also might contribute to impaired synaptic plasticity in Mecp2−/y KO mice as demonstrated in conjunction with long-term potentiation in the hippocampus (
;
). Our results indicate a lack of desensitization of the HBR in KO mice and also suggest disturbed plasticity within the respiratory network. Certain forms of short-term synaptic plasticity within the respiratory network undergo substantial maturation (
). A disturbed maturation of these network circuits in RTT might therefore particularly affect plasticity dependent adaptive behaviour of the respiratory network.
Conclusions
The main finding of our study is a dysfunction of the central and vagal control of the postinspiratory activity in Mecp2−/y KO mice. The results are of clinical interest because most of the respiratory abnormalities in patients with RTT including repetitive apnoeas, breath-holding spells, Valsalva's manoeuvres and loss of speech can potentially be explained by similar pathophysiological mechanisms, which are the main basis for devising effective pharmacological therapies.
Acknowledgments
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, Göttingen, Germany). The authors would like to thank A. Bischoff for excellent technical assistance, E. Munk for animal breeding and husbandry, and K. Köhler (Department of Genetic Epidemiology, Georg August University Göttingen) for statistical advice.
Disclosure
The authors have reported no conflicts of interest.
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