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1Department of Obstetrics and Gynecology and 2Department of Cell and Development Biology, Oregon Health and Sciences University, Portland, Oregon
Submitted 6 August 2007 ; accepted in final form 12 November 2007
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ABSTRACT |
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control of respiration; Rett syndrome
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METHODS |
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The protocols used were approved by the Oregon Health and Sciences Institutional Animal Care and Use Committee and were in agreement with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Heterozygous (Mecp2+/–) and wild-type (Mecp2+/+) females were obtained by crossing B6.129P2(C)-Mecp2 tm1.1Bird (stock number: 003890; Jackson Laboratory, Bar Harbor ME) heterozygous females with C57BL/6J males. Mice were genotyped using the protocol of the supplier. This strain was originally generated by insertion of loxP sites around Mecp2 exons 3 and 4 and crossing homozygous floxed females with CMV Cre males (13).
Under general inhalation anesthesia (1.5% isoflurane in oxygen), a midline abdominal incision was made, and the liver was gently retracted. A ligature was passed around the esophagus at its entrance to the stomach, and the stomach was retracted to allow visualization of the lower esophagus. A 25-gauge hypodermic needle was used to make an incision in the longitudinal muscle of the esophagus, and with a blunt probe a tunnel was made between the outer and inner layers of the muscle. This tunnel extended beyond the diaphragm into the thoracic cavity. The tip of a catheter attached to a pressure transducer (model PA-C10, Data Sciences International, St. Paul, MN) was advanced into the thoracic cavity and secured by gluing it to the entry point in the longitudinal muscle. The suture rib of the pressure transducer was incorporated into the abdominal musculature closure and the skin approximated with a subcuticular suture. This surgical procedure was adapted from published reports (15, 21). Mice were allowed 5–7 days to recover before any studies were performed.
Experimental Protocols
Observation. In this protocol mice were studied in their home cages for 2–3.5 h to determine their baseline respiratory pattern. The cage was placed on a telemetry receiver (model RBC-1, Data Sciences), and pleural pressure was continuously recorded. These studies were conducted between 1800 and 2130 in animals that were on a 12:12-h light-dark cycle with lights on from 0600 to 1800.
Oxygen or carbon dioxide administration. For these studies mice were placed in a 2-liter clear Plexiglas chamber with access to food and water. The chamber had intake and output ports for flow-through administration of respiratory gases. Animals were exposed in random order to room air; 12% oxygen/88% nitrogen; 40% oxygen/60% nitrogen; or 2% carbon dioxide in air, each for 30 min.
Data Analysis
Pleural pressure waveforms were sampled at 2 kHz, and breath intervals were calculated from the peak negative deflections. Respiratory frequency and breath amplitude were determined using custom-written functions in Igor Pro 5.04 B (WaveMetrics, Lake Oswego, OR). Periodic breathing was defined as an episode of three or more cycles of 3–30 breaths, separated by respiratory pauses (Figs. 2 and 4). A respiratory pause was defined by a breath interval equal or greater than twice normal intervals. For each episode of periodic breathing, the period (time from midpoint of one breath cycle to the next breath cycle), the length of the episode, and the ventilation:apnea ratio were calculated. Ventilation:apnea was determined for each episode and averaged for the 30-min trial. The ratio was obtained by dividing the time in which breathing was present by the time occupied by absence of respiratory activity. Animal activity was measured with the telemetry system. As the mouse moves, the telemetry signal transmitted to the receiver antennas varies in strength. Changes in signal strength generate an activity count that is dependent on both distance and speed of movement. Activity was recorded as either inactive (no counts) or active (2–160 counts/min).
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Results are given as means ± SE. Single comparisons such as number of periodic breathing epochs per hour in the observation protocol between Mecp2+/+ and Mecp2+/– mice were made with unpaired Student's t-test. The effects of oxygen concentration were evaluated with two-way repeated-measures ANOVA, with strain and oxygen concentration as the two factors. P < 0.05 was considered significant.
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RESULTS |
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Mecp2+/+ (n = 10) and Mecp2+/– (n = 11) mice were grossly indistinguishable. Their weights were similar, 23.0 ± 0.7 and 23.5 ± 8 g. Brain weights in Mecp2 deficient mice (418 ± 7 mg) were less than wild-type (448 ± 6 mg, P = 0.013). Ten of eleven Mecp2+/– animals clasped their hind legs when elevated by the tail (13).
Observational Study
Analysis of respiratory frequency during 2- to 3.5-h periods showed a bimodal distribution. Activity measurements in five Mecp2+/+ and six Mecp2+/– mice determined that the rapid breathing occurred when the animal was active and the slower frequency occurred while the animal was inactive. Mecp2+/– mice breathed slower than Mecp2+/+ mice while inactive (2.7 ± 0.1 vs. 4.4 ± 0.5 Hz, P = 0.019) and during active periods (6.4 ± 0.3 vs. 8.5 ± 0.5 Hz, P = 0.008). Periodic breathing was characterized by a waxing and waning pattern in breath amplitude (Figs. 2 and 4). A number of the periodic breathing episodes were immediately preceded by a large-amplitude breath or sigh: 61.5 ± 4.3% for Mecp2+/– and 41 ± 7.5 for Mecp2+/+ (e.g., normoxia in Fig. 4). Episodes of periodic breathing occurred 9.4 ± 2.2 times per hour in heterozygous females compared with 2.5 ± 0.4 in wild-type (P = 0.01). In addition, the epochs were longer in Mecp2 deficient animals than in wild type (15.3 ± 1.7 vs. 8.6 ± 0.7 s, P = 0.0003).
The incidence of periodic breathing was not affected by mouse age at the time of study (Fig. 1). Mecp2+/– animals comprised two populations. Five of the 11 mice had a frequency of periodic breathing within the range seen in wild-type mice, while the other six were outside this range (Fig. 1). The two groups of Mecp2+/– mice did not differ with respect to the average length of their periodic breathing episodes (13.4 ± 2.2 vs. 14.8 ± 1.7 s) or to the periodicity of their cycles (2.4 ± 0.2 vs. 2.5 ± 0.4 s). The six Mecp2+/– mice with a high incidence of periodic breathing, however, showed a lower ventilation:apnea ratio compared with the other five (2.06 ± 0.23 vs. 2.78 ± 0.18, P = 0.042), indicating a higher loop gain (9).
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The effect of breathing varied oxygen concentrations was studied in six wild-type mice. Oxygen concentrations did not affect the total amount of time in periodic breathing in Mecp2+/+ mice (Fig. 3A) (P values between 0.508 and 0.613). Exposure to 40% and 12% oxygen did not change the number of periodic breathing episodes: 4.3 ± 1.0 and 0.8 ± 0.40, respectively, compared with 4.7 ± 1.80 per 30 min in air (40% vs. 12%, P = 0.177; 12% vs. air, P = 0.177) (Fig. 3B). Relative hypoxia eliminated periodic breathing in three of six wild-type mice (Fig. 3B). The length of these episodes was also not affected: 10.0 ± 2.4 s in 21%, 7.3 ± 1.1 s in 12%, and 7.4 ± 1.1 s in 40% oxygen (P values between 0.209 and 0.878) (Fig. 3C). The ventilation:apnea ratio fell significantly from 5.17 ± 0.50 in hypoxia to 3.73 ± 0.28 and 3.5 ± 0.28 in normoxia and hyperoxia, respectively (P = 0.027 and 0.035) (Figs. 2 and 3D).
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The effects of varied oxygen concentrations on periodic breathing were not secondary to animal activity while breathing the three mixtures. Heterozygous females were active 45 ± 9.2% of the time in air, 36 ± 4.5% in hypoxia, and 42.3 ± 6.3% in hyperoxia (P = 0.51). Similarly, wild-type mice were active 38.5 ± 3.5, 63.2 ± 10.8, and 67.5 ± 12.9% in room air, 12% oxygen, and 40% oxygen, respectively (P = 0.25).
Effect of Carbon Dioxide
In separate experiments the incidence of periodic breathing was studied in Mecp2+/– mice breathing 2% carbon dioxide-98% air compared with room air. Carbon dioxide reduced the total amount of periodic breathing from 103 ± 15 to 14 ± 3 s/30 min (P = 0.046). Both the number of episodes (5.7 ± 1.8 vs. 1.3 ± 0.2) and the average length of the episodes (13.8 ± 1.2 vs. 9.2 ± 0.9 s) were less when breathing 2% carbon dioxide.
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DISCUSSION |
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Strohl and coauthors (14, 33) have shown that periodic breathing in mice is affected by strain. C57BL/6J mice demonstrated periodic breathing after short (1 or 5 min) poikilocapnic hypoxia (8% oxygen) when they were returned to 100% oxygen. In contrast A/J animals did not show this respiratory instability (14). The authors attribute the differences in part to the fact that A/6 mice have short-term potentiation of ventilation after hypoxia that promotes stability, while C57BL/6J animals show posthypoxic frequency decline that contributes to periodic breathing. On recovery from 5 min of poikilocapnic (8% oxygen) hypoxia, heterozygous Mecp2 deficient mice showed a greater decline in ventilation than wild type (5). This may contribute to their greater prevalence of periodic breathing. Previous studies of C57BL/6J animals did not observe spontaneous episodes of periodic breathing (14). The experimental conditions were different from those reported here. Han et al. (14) used whole body plethysmography and studied animals between 1000 and 1400, whereas our observations were made with telemetry between 1800 and 2130 with the mice in their home cages.
Recently Strohl and associates (33) have extended their studies of periodic breathing in C57BL/6J mice. A 1-min exposure to 8% oxygen followed by recovery in 100% oxygen resulted in periodic breathing in all nine mice treated with vehicle. In contrast, the carbonic anhydrase inhibitor acetazolamide eliminated periodic breathing in all nine animals. Since acetazolamide caused a decrease in hypercapnic ventilatory response without affecting either poikilocapnic or isocapnic hypoxic responses in these mice, the authors conclude that respiratory instability under these conditions is caused by mechanisms that impinge on or are within the central respiratory controller. As discussed below, this is consistent with the mechanisms we suggest for increased periodic breathing in heterozygous Mecp2 deficient females. It should be noted that periodic breathing in these C57BL/6J animals is always induced during hyperoxic recovery from a brief hypoxia, while the instability seen in Mecp2+/– occurs spontaneously in normoxia.
Previous studies in Mecp2 heterozygous female mice (5) and Mecp2 null males (25, 29, 32) have not reported this pattern of instability. The females were studied with body plethysmography in which they were restrained by a close fitting hole in Parafilm about their head. These studies lasted less than 1 h and were designed to measure acute responses to hypoxia or hypercapnia (5). The stimulation induced by restraint may have precluded observation of periodic breathing. Null males were examined either in whole body plethysmography (25, 32) or using a perfused working heart-brain stem preparation (29). In the whole body studies, records were analyzed only when the mice were without limb, body, or head movement. Since they may well have been asleep at the time (30), this may explain the difference. In the perfused working heart-brain stem experiments (29), the lungs are removed, thus eliminating the pulmonary afferents that were present in our studies. As periodic breathing episodes are often preceded by an augmented breath (see RESULTS), pulmonary afferents may contribute to this respiratory pattern.
Periodic breathing has for some time been examined in terms of the engineering concept of loop gain (18, 19, 34). Loop gain is the ratio of the response in ventilation to that of the initial change in ventilation that produced a change in alveolar gas tensions. The changes in gas tension are sensed by chemoreceptors (central and peripheral) that in turn result in the ventilatory response. Loop gain has three components. 1) The first component is plant factors, those factors that determine the extent that pulmonary blood gas tensions will change for a given change in minute ventilation, such as functional residual capacity. 2) The second component is gain imposed by mixing of alveolar capillary blood with that in thoracic vessels and both circulatory delays from lung to peripheral and central chemoreceptors and diffusion delays in reaching central chemoreceptors. Heterozygous Mecp2 deficient females have autonomic cardiovascular regulation that is similar to wild type (6). Cardiac output and circulatory times, however, have not been determined. 3) The third component is controller gain. These include sensitivity of peripheral and central chemoreceptors to changes in blood gas tensions and the response of premotor and motoneurons to input from these chemoreceptors. As mentioned in the introduction, the ventilatory response to hypoxia is greater in Mecp2+/– mice compared with Mecp2+/+ (4, 5). The failure of relative hyperoxia to correct periodic breathing in the heterozygotes, however, argues against increased peripheral chemoreceptors underlying their respiratory instability.
In humans at altitude not all individuals demonstrate periodic breathing during sleep. The ventilatory response to hypoxia was considerably larger in periodic breathers compared with those with stable breathing (20). Similarly, in periodic breathing induced by hypoxia coupled with a breathing circuit that allowed augmented inspired oxygen concentrations, only a subset of subjects had instability. Periodic breathers had greater responses to both hypoxia and hypercapnia (7). In addition, supplemental oxygen suppresses periodic breathing at natural altitude (20) and that seen in an altitude chamber (3). Based on this background and the findings that Mecp2 deficient mice have an augmented hypoxic response (4, 5), it was anticipated that relative hyperoxia would relieve, not worsen, periodic breathing. Relative hypoxia eliminated periodic breathing in a number of wild-type and heterozygous Mecp2 deficient females. This is consistent with the carotid body contributing to respiratory stability in mouse. Addition of 2% carbon dioxide significantly diminished periodic breathing in heterozygous Mecp2 deficient female mice. This response is similar to that seen in humans (3) and animals (9) and suggests that the respiratory pauses in periodic breathing are associated with hypocapnia.
The ventilation:apnea ratio in Mecp2+/– mice varied inversely with inspired oxygen concentration (Fig. 5D). Increased apnea duration in periodic breathing reflects a greater plant gain. In animals that were ventilated at increased respiratory frequencies and tidal volume to induce periodic breathing, larger tidal volumes resulted in a fall in ventilation:apnea ratio (9). This mechanical increase in plant gain did not alter the period length during the unstable breathing. This is the same result we have seen with oxygen administration, suggesting that relative hyperoxia increases plant gain. This effect of hyperoxia coupled with the increased total amount of periodic breathing and number of episodes compared with normoxia is consistent with the conclusion that peripheral chemoreceptors contribute to respiratory stability in Mecp2 deficient mice.
As opposed to peripheral chemoreceptors underlying periodic breathing in the Mecp2 deficient mice, a central mechanism is suggested. Respiratory neurons have inhibitory inputs that occur concurrently with stimulation during inspiratory bursts. Extracellular potentials recorded from medullary ventral respiratory group and dorsal respiratory group neurons before and after iontophoretic application of bicuculline demonstrated that the GABAA receptor antagonist produced a marked increase in discharge (11, 27). Similarly, in a neonatal rat isolated brain stem-spinal cord preparation, bicuculline resulted in an increase in the integrated phrenic motoneuron (C4) burst envelope (23). Immunoblots of whole adult mouse brain showed that Mecp2+/– mice have only 60–70% of the wild-type expression of the β3 subunit of the GABAA receptor (26). Complete lack of β3 subunit severely impairs inhibition in a number of neuronal circuits. Mice lacking β3 have a 50% reduction in the frequency and amplitude of spontaneous inhibitory postsynaptic currents (16), in the thalamic reticular nucleus. Application of the GABAA agonist muscimol to granular cells of the olfactory bulb produced much reduced currents in these β3 null animals compared with wild type (22). A reduced GABAergic inhibition to inspiratory bursts in Mecp2+/– mice would result in an increased controller gain that may underlie their breathing instability. It is not known whether oxygen tension modulates the GABAergic input to phrenic and medullary neurons during inspiration. GABA does increase rapidly during hypoxia (24) and therefore could modulate central drive, resulting in the decrease in periodic breathing observed.
There are, nonetheless, reports that argue against this proposed mechanism. Whole cell electrophysiological recordings from layer 5 pyramidal neurons in slices from the somatosensory cortex of Mecp2 null male mice (–/y) found that spontaneous firing was reduced in Mecp2–/y compared with wild type (10). The difference was not due to the intrinsic excitability of the neurons. Rather, the average excitatory synaptic charge was decreased and the average inhibitory synaptic charge increased in Mecp2 null males. Recent preliminary results from the same laboratory, however, have shown that noradrenergic neurons in the locus ceruleus of Mecp2–/y animals have a firing frequency that is 1.7 times that of Mecp2+/y (31). It may well be that Mecp2 deficiency has opposite effects in the brain stem compared with the cortex.
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GRANTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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ACKNOWLEDGMENTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSACKNOWLEDGMENTSREFERENCES |
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This article has been cited by other articles:
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  C. Gaultier and J Gallego Neural control of breathing: insights from genetic mouse models J Appl Physiol, May 1, 2008; 104(5): 1522 - 1530. [Abstract] [Full Text] [PDF]
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