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J Appl Physiol 103: 552-559, 2007. First published May 24, 2007; doi:10.1152/japplphysiol.01389.2006
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Altered respiratory pattern and hypoxic response in transgenic newborn mice lacking the tachykinin-1 gene

J. Berner,1,2 Y. Shvarev,1,4 H. Lagercrantz,1 A. Bilkei-Gorzo,3 T. Hökfelt,2 and R. Wickström1

Departments of 1Woman and Child Health and 2Neuroscience, Karolinska Institutet, Stockholm, Sweden; 3Department of Molecular Neurobiology, University Hospital Bonn, Bonn, Germany; and 4Neurogenomic Laboratory, Institute of Cytology and Genetics, Siberian Division of the Russian Academy of Sciences, Novosibirsk, Russia

Submitted 7 December 2006 ; accepted in final form 15 May 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Substance P is known to be involved in respiratory rhythm and central pattern-generating mechanisms, especially during early development. We therefore studied respiratory responses in transgenic newborn mice (Tac1–/–) lacking substance P and neurokinin A (NKA). In vivo, the effects of intermittent isocapnic hypoxia (IH) and hypercapnia were studied using whole body flow plethysmography at P2-3 and P8-10. In vitro, anoxic responses and the effects of hypocapnic and hypercapnic conditions were studied in brain stem-spinal cord preparations (C4 activity) at P2. Hypoxic challenge considerably modified the respiratory activity in transgenic mice displayed in vivo as an attenuated increase in tidal volume during IH. Transgenic mice also showed a more prominent posthypoxic frequency decline in vivo, and posthypoxic neuronal arrests appeared more often in vitro. We recognized two types of sigh activity: with or without a following pause. During IH, the amount of sighs with a pause decreased and those without increased, a redistribution that became stronger with age only in controls. Intermittent anoxia induced long-term facilitation effects in controls, but not in Tac1–/– animals, manifested as an increase in burst frequency in vitro and by an augmentation of ventilation during posthypoxic periods in vivo. Thus our data demonstrate that a functional substance P/NKA system is of great importance for the generation of an adequate respiratory response to hypoxic provocation in newborn mice and during early maturation. It also indicates that substance P (and/or NKA) is involved in the development of the plasticity of the respiratory system.

augmented breath; apnea; neonatal; sudden infant death syndrome

THE RESPIRATORY CONTROL SYSTEM is dependent on a sophisticated balance and specific participation of different neuromodulator systems. Thus the respiratory control system must be stable and yet respond to different stimuli like emotions, exercise, speech, and diseases. Substance P is considered to be one of the neuromodulators involved in this complex and finely tuned system (8, 31, 38, 78, 83). It is an undecapetide belonging to the tachykinin family also comprising neurokinin (NK) A, NKB, neuropeptide K, and neuropeptide {gamma}. All tachykinins are encoded by the Tac1 gene in mouse, except for NKB, which is encoded by the Tac2 gene. Substance P preferentially acts via NK-1 receptors, whereas NKA and NKB interact with NK-2 and NK-3 receptors, respectively (63, 65, 67). Substance P is one of the most abundant and widespread neuropeptides in the central and peripheral nervous system and is involved in several physiological processes, including cardiovascular, respiratory, gastrointestinal, and nociceptive processes, and also modulates the immune response (24, 33, 48, 55). It is present in sensory afferents from arterial baroreceptors and chemoreceptors and is also found in areas in the brain stem thought to be involved in respiratory regulation (19, 25, 27, 35, 36). One of the areas is the ventrolateral medulla, including the pre-Bötzinger complex, which is assumed to generate respiratory rhythms as eupnic breathing, sighing, and gasping (23, 34, 69, 70). A selective lesion of NK-1-expressing neurons in the pre-Bötzinger complex results in a profoundly abnormal respiratory pattern (22, 76, 77), which indicates a vital role for these substance P-sensitive neurons in generating a normal respiratory response.

The respiratory system undergoes crucial changes during the perinatal period, and substance P may play an important role during this maturation (50, 58, 60, 79, 80, 83). Attention to breathing pattern recognition has grown in recent years as the idea of the plasticity of the neural network has emerged (15, 34, 46). This developmental plasticity of respiratory control is especially vulnerable during this critical period (for review, see Refs. 7, 21, 61), and intermittent hypoxia has been shown to cause a structural and functional modification of the central nervous system even after the impact has ceased (62). This modulation, referred to as long-term facilitation (LTF), is considered an adaptive mechanism adjusting breathing to different environmental factors (5, 29, 53, 56). Moreover, human infant victims of sudden infant death syndrome (SIDS) have elevated levels of substance P in medulla oblongata (4, 43, 49), and substance P is decreased in the brain stem in Rett syndrome patients suffering from an abnormal breathing pattern, including apneas, during early development (11, 66).

We therefore wanted to study the effect of intermittent hypoxia on newborn animals lacking the Tac1 gene. By combining in vivo and in vitro methods, we are able to estimate the relative contribution of respiratory effects at different levels. The objectives of this study were thus to determine 1) the intermittent hypoxic and hypercapnic respiratory response and developmental changes, 2) whether substance P/NKA is involved in the plasticity of respiratory network, with and without peripheral input, and 3) alterations in central chemosensitivity in newborn mice lacking the Tac1 gene.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals.   Homozygous Tac1 gene deleted mice of both sexes were used. The Tac1–/– deleted mouse line was generated from C57/BL6J and kindly provided by Dr. Andreas Zimmer (84). C57/BL6J mice pups of both sexes (B & K Universal, Sollentuna, Sweden) were used as controls. The transgenic mice were viable and fertile. All pups were born naturally and left with their mothers until assigned age. Animals were kept on a regular 12-h light-dark cycle and treated according to the guidelines approved by the local ethics committee (see below). The pups were studied at either postnatal day 2 or 3 (P2-3) or at postnatal day 8, 9, or 10 (P8-10). In all groups, pups came from at least three different litters and were only tested once.

In vivo protocols and ventilatory measurements.   Ventilatory measurements were made using noninvasive, whole body flow plethysmography. All studies were performed between 0900 and 1500 to control for possible daily fluctuations in respiratory patterns. Pups were placed in a plethysmograph chamber (MTA, Karolinska University Hospital) in which they could move freely (the chamber volume was 35 ml for newborn mice and 100 ml for the older age group). The airflow through the box was maintained at 100 ml/min, and gases were exchanged using a computer controlled valve switch. Control experiments verified that a 95% gas exchange had occurred after 30 s. The chamber was connected to a highly sensitive direct airflow sensor (TRN3100, Kent Scientific, Litchfield, CT), and the signal was amplified with a four-channel amplifier (PN 770 S/N5, Somedic Sales, Hörby, Sweden). The signal was digitally converted with a sampling rate of 100 Hz and analyzed using Dasylab software (Datalog, Mönchengladbach, Germany). The obtained values were then used to calculate respiratory frequency (f; breaths/min), tidal volume (VT; µl), and ventilation (VE; ml/min). VT and VE were divided by body weight (g) and expressed as microliters per gram and milliliters per gram per min, respectively. The method used is a completely noninvasive and unrestrained approach, which includes refraining from measuring rectal temperature. Since the open-chamber, flow-through system registers amplitude differences in the outgoing airflow, which are due to several factors (14), the setup does not allow determination of absolute tidal volumes. We therefore only compare relative values between animals and groups. Ambient temperature within the chamber was measured continuously using a digital thermometer and recorded in parallel with the flow signal. It was maintained at 33 ± 0.5°C in accordance with the thermoneutral range for mice of similar age by immersing the chamber in a thermostat-controlled water bath (39).

Animals were kept 3 min in the chamber in room air for acclimatization before recording started. In the hypoxic protocol, eight animals in each group at P2-3 and nine animals in each group at P8-10 [wild-type (WT) group and transgenic group] were included. They were exposed to normoxia (21% O2) for 3 min (baseline values), followed by three episodes of IH and mild hypercapnia (8% O2, 3% CO2). The hypoxic mixture contained 3% CO2 to maintain near-normal arterial PCO2 values during hypoxia (10, 45, 54). Each isocapnic hypoxic period lasted 5 min followed by a normoxic recovery period of 2 min. In the hypercapnic protocol, 9 animals in each group at P2-3 and 10 animals in each group (WT group and transgenic group) were included. This protocol also included 3 min of normoxia, to obtain baseline values, followed by 15 min of moderate hypercapnia (5% CO2, 21% O2).

The pups were weighed after the final recording. Analysis of the respiratory signal was made on 30-s segments without movement artifacts that were chosen subjectively within assigned intervals. For basal respiration, this interval was during minute 3 of air (basal respiration) and for each hypoxic period during minutes 2, 3, and 5. During hypercapnia, minutes 3, 6, 9, 12, and 15 were selected. The results of the respiratory measurements were used for comparative analyses only (14). The number of apneas (defined as ventilatory pauses longer than twice the duration of the preceding breaths) was calculated manually, as were sighs or augmented inspiratory breaths [AIB; defined as biphasic inspiration, twice the VT of the preceding breaths (9, 74)]. Two different types of AIB were observed, one type with a following pause (47) and another type of AIB characterized as an incomplete sigh with an inspiratory phase of more than twice the amplitude of preceding breaths, immediately followed by an expiration but without a following pause (16) (Fig. 2, A and B). All measurements were made in at least 15-s-movement artefact-free sequences.


Figure 1
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  		Fig. 1. The respiratory response to intermittent isocapnic hypoxia (IH) in mice at P2-3 (left) and P8-10 (right). Animals at P2-3 display an immature breathing frequency (f) response with an immediate decrease but a tendency to reach above baseline during posthypoxic periods that becomes significantly increased in second and third posthypoxic period (A; *P = 0.039 and P = 0.043, respectively). In contrast, f increases similarly in both groups at P8-10, but during posthypoxic periods transgenic mice display a significantly decreased f compared with basline (B; *P = 0.036, P = 0.004, P = 0.027 in first, second, and third hypoxic period, respectively). Furthermore, tidal volume (VT) is unaltered at P2-3 (C), whereas a significant increase is seen in wild-type (WT) animals at P8-10 (D) during IH compared with baseline (P < 0.01 in first and second hypoxic period, P < 0.03 in third). However, transgenic animals retain the immature VT response compared with WT (F; *P = 0.005, P = 0.008, P = 0.017 in first, second, and third hypoxic period, respectively). Thus the increase in minute ventilation (VE) in response to IH seen in WT animals at P8-10 is obliterated in transgenic animals (F; *P = 0.014, P = 0.044, P = 0.040 in first, second, and third hypoxic period, respectively). Significant differences in VT and VE during posthypoxic periods (air) in age group P8-10 are seen in first and third period between the experimental groups (D: §P = 0.027 and §P = 0.008, respectively; and F: §P = 0.039 and §P = 0.013, respectively). Please note the different scales between f, VT, and VE. Vertical bars denote means ± SE. bw, Body weight.

 

Figure 2
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  		Fig. 2. The different distribution of augmented inspiratory breaths (AIB) activity during intermittent isocapnic hypoxia in age groups P2-3 and P8-10. WT transforms toward AIB without pause in response to intermittent hypoxia, which becomes stronger with age. A: an example of an AIB with pause. B: AIB without pause (processed data from the plethysmography). C and E: decreased AIB with pause activity in WT. In P2-3, it becomes significant in the second and third hypoxic period compared with Tac1–/– mice (*P = 0.036 and P = 0.0013, respectively). In age group P8-10, the decrease in WT becomes even more obvious and is significant in all three hypoxic periods (*P = 0.002, *P = 0.013, and *P = 0.009, respectively) compared with Tac1–/– mice. D and F: AIB without pause. The transient increase seen at P2-3 in both experimental groups becomes stronger with age, and at P8-10 WT display a significant increase in first hypoxic period compared with Tac1–/– mice (*P = 0.030). Vertical bars denote means ± SE. Please note different scaling between C and D, and E and F.

 
Brain stem-spinal cord preparation.   Experiments were performed on the brain stem-spinal cord preparation of 2-day-old (P2) pups. The isolated brain stem and cervical spinal cord were obtained under isoflurane anesthesia as described previously (42). The brain stem was then rostrally cut between the sixth cranial nerve roots and the lower border of the trapezoid body and caudally between sixth and seventh spinal nerves. The isolated preparation was continuously perfused at the rate of 3.5–4.5 ml/min in a 2-ml chamber with a solution containing (in mM) 130 NaCl, 5.4 KCl, 0.8 KH2PO4, 0.8 CaCl2, 1 MgSO4, 26 NaHCO3, 30 glucose and equilibrated with 95% O2 and 5% CO2 at 28°C; pH was set at 7.4 [control artificial cerebrospinal fluid (ACSF)].

In vitro protocols, data recording, and analysis.   Extracellular recordings monitored inspiratory discharges of respiratory motoneurons with glass suction electrodes applied to the proximal cut end of the C4 ventral roots of the spinal nerves. The burst frequency and amplitude were analyzed. The burst frequency was calculated as a number of C4 bursts per minute. The respiratory C4 burst amplitude was analyzed in integrated and smoothed ({tau} = 50 ms) recordings. Burst amplitude was quantified (arbitrary units) as the distance from the baseline to the peak of the integrated burst slope. The control values of the inspiratory burst frequency and amplitude were calculated during application of normoxic and normocapnic ACSF as the mean of the last 3 min before testing ACSF application. During hypocapnic and hypercapnic conditions, the burst frequency and amplitude were calculated as the mean of the last 3 min of ACSF application. During anoxia, the burst frequency and amplitude were calculated as a mean over 1 min when the maximal effect was observed. Axoscope software and Digidata 1200B interface (Axon Instruments, Foster, CA) were used to collect electrophysiological data. Solution PO2 and PCO2 in the chamber were measured using an ALB 505 analyzer (Radiometer, Copenhagen, Denmark). Offline analysis was performed employing Clampfit 8.02 (Axon Instruments), DATAPAC 2K2 (RUN Technologies, Laguna Hills, CA), Statistica 6 (Stat Soft, Tulsa, OK), Sigma Stat (SPSS, Chicago, IL), and Origin 6.0 (Microcal Software, Northampton, MA) software for PC.

After the preparation was superfused with control ACSF (with PO2 of ~85 kPa and PCO2 of ~5 kPa) for 40 min and C4 activity reached a steady state, the control perfusate was replaced by testing solutions. In protocol 1, control ACSF was replaced for 25 min by hypocapnic solution (pH 7.2, PO2 of ~85 kPa and PCO2 of ~2 kPa) equilibrated with 92% O2, 6% N2, and 2% CO2, followed for the next 25 min by a hypercapnic solution (pH 7.8, PO2 of 85 kPa, and PCO2 of ~8 kPa) equilibrated with 92% O2 and 8% CO2. Then, after washing the preparations with control ACSF for another 30 min, the anoxic solution (PO2 of ~15 kPa, PCO2 of ~5 kPa) equilibrated with 95% N2 and 5% CO2 was applied for 15 min followed by control ACSF for another 40 min. Supplementary sessions were designed to estimate possible effect of preliminary exposure to hypocapnic and hypercapnic ACSF on anoxic response in WT and Tac1–/– prepartions. For this, in protocol 2, anoxic ACSF was applied to the preparations at the same period of time as in protocol 1 but without exposure to hypocapnic and hypercapnic ACSF. In protocol 3, after initial 40-min adaptation, the intermittent anoxia consisting of three 3-min intervals of anoxia separated by 5 min of normoxia was applied to the preparations. The last anoxic episode was followed by a 40-min interval of washing out with the control ACSF.

Statistical considerations.   Statistical analysis of baseline values (displayed in Table 1) was performed by Student's t-test, as were other paired comparisons. For multiple statistical comparisons, one- and two-way ANOVA (Statistica software, Statsoft Scandinavia, Uppsala, Sweden) designs for repeated or independent measurements were used where appropriate. For respiratory parameters, repeated-measures ANOVA were used with WT, transgenic mice, and gender as between factors and repeated hypoxic exposure (at 3–8, 10–15, and 17–22 min) or hypercapnic exposure (measured at minutes 3, 6, 9, 12, 15) as the within-subjects factor. Post hoc analysis was conducted by the Tukey's test. Statistical analysis of respiratory pattern (i.e., sighs, apneas, and AIB) was performed using the Mann-Whitney's U-test. All statistical analyses were made on absolute values before normalization for graphical clarity. Data are shown as means ± SD and means ± SE in figures for graphical clarity. Unless stated otherwise, all P values refer to two-way ANOVA. Significance level was set at P < 0.05.


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  		Table 1. Baseline values during normal conditions in room air

 
Ethical considerations.   This experiment has been approved by the local ethical committee, i.e., Stockholms norra djurförsöksetiska nämnd (Dnr N88/02 and 166/04).


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
In vivo.   During basal conditions, transgenic mice in age group P2-3 displayed significantly less apneas compared with WT mice. However, there were no differences in f, VT/body wt or VE/body wt between groups at any age. Similarly, no differences were found in the frequency of AIB of any type in either age group. The body weight (g) was significantly higher in transgenic mice than in WT in both age groups. Basal conditions are summarized in Table 1.

Intermittent isocapnic hypoxia.   In age group P2-3, no clear respiratory response to isocapnic IH was seen (Fig. 1, A, C, and E). At P8-10, VT/body wt increased significantly more in WT compared with Tac1–/– mice, whereas both experimental groups displayed a similar increase in f during isocapnic IH. Transgenic mice thus retained their immature response (Fig. 1D). Consequently, a significant attenuation of net ventilation (VE/body wt) was seen in transgenic mice compared with WT (Fig. 1F). During the normoxic periods following each hypoxic challenge, f increased in both experimental groups at age P2-3 (second and third posthypoxic period; see Fig. 1A). In contrast, in transgenic mice at age P8-10, the f was significantly depressed compared with baseline, whereas in the WT group f returned close to baseline (Fig. 1B). Furthermore, both VT/body wt and VE/body wt were significantly depressed below baseline in transgenic animals compared with WT that never returned to baseline levels (Fig. 1F).

The number of apneas per minute, seen during normal conditions (P2-3), remained significantly less in transgenic mice during isocapnic IH (P = 0.010, P = 0.024, P = 0.036 in first, second, and third posthypoxic period, respectively; data not shown). This difference disappeared with age, when apneas in WT decreased to a very few, as expected at P8-10.

The total AIB activity (i.e., with and without pause combined) increased in both groups during the first hypoxic period and then declined in WT but remained high in Tac1–/– mice at age P2-3 (P = 0.018 in third hypoxic challenge compared with WT; data not shown). At age P8-10, there was a similar tendency, but this did not reach statistical significance (P = 0.20 in third hypoxic period).

The distribution of the two types of AIB was different between the experimental groups in response to intermittent isocapnic hypoxia, where the amount of sighs with a pause decreased and those without increased, a redistribution that became stronger with age in controls but not in transgenic mice (Fig. 2, C–F).

Hypercapnia.   The hypercapnic ventilatory response was similar between experimental groups at both ages. Likewise, there were no differences between WT and Tac1–/– mice (in either age group) in the frequency of AIB (with or without pause) or apneas when challenged with moderate hypercapnia.

In vitro.   Respiratory activity was monitored throughout the whole recording session in control experiments to estimate the stability of the preparation since conditions may change during longer experiments. In control (normoxic) conditions, the basic burst frequency was similar in both experimental groups: 5.85 ± 1.3 bursts/min (n = 16) in WT vs. 5.01 ± 1.2 bursts/min (n = 18) in Tac1–/–. Hypocapnic and hypercapnic ACSF significantly and equally affected the burst frequency in WT (n = 9) and Tac1–/– (n = 10) preparations, decreasing it by 86–91% of control values during hypocapnia (P < 0.001) and increasing it by 129–133% during hypercapnia (P < 0.001).

In both transgenic and WT mouse brain stem preparations, the anoxic response was characterized by an initial transient increase in burst frequency and a reduction in burst amplitude. The burst amplitude was subsequently restored, and the bursting continued throughout anoxic conditions with a significantly decreased frequency (Fig. 3). Moreover, resumption of oxygenation induced appearance of posthypoxic neural arrest (PHNA) in some preparations.


Figure 3
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  		Fig. 3. C4 activity in brain stem-spinal cord preparations (P2). Effects of continuous anoxia (15 min) on respiratory output in brain stem preparations in WT and Tac1–/– mice. PHNA, posthypoxic neuronal arrest during resumption of oxygen.

 
There were no differences between groups in anoxic frequency response, which was also independent of the preliminary exposure of the WT (n = 6) and Tac1–/– (n = 8) preparations to hypocapnic-hypercapnic conditions. During prolonged anoxia, the frequency increased up to 242 ± 24.7% in WT and 254 ± 46.5% in Tac1–/– mice (P < 0.001). However, the burst amplitude reduction during the anoxic response was significantly less in Tac1–/– mice, decreasing to 51.4 ± 18.4% (n = 16) of control values in transgenic mice vs. 19.5 ± 12.3% in WT (n = 18) (t-test, P < 0.001). The duration period when the burst amplitude recovered in the case of prolonged anoxia was also significantly shorter in Tac1–/– mice compared with WT mice: 234.74 ± 100.5 vs. 328.4 ± 61.4 s (t-test, P < 0.05). Furthermore, PHNA following resumption of oxygenation appeared significantly more often in Tac1–/– preparations, and it was observed in 31% (5 of 16) of WT and in 83% (15 of 18) of Tac1–/– preparations (z-test, P < 0.001).

The changes in the inspiratory burst frequency were also very similar in Tac1–/– and WT preparations during intermittent anoxic challenge (P > 0.05; Fig. 4; Table 2). After intermittent anoxia, PHNA was seen in 43% (3 of 7) of WT and 100% (10 of 10) of Tac1–/– preparations (Fig. 4; z-test, P < 0.05).


Figure 4
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  		Fig. 4. Effects of intermittent anoxia on respiratory output in brain stem preparations in control (WT) and Tac1–/– mice. The intermittent anoxia consisted of three 3-min intervals of anoxia separated by 5 min of normoxia. Posthypoxic neuronal arrest (PHNA) was observed in 100% of Tac1–/– preparations due to intermittent anoxia compared with 43% in control preparations.

 

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  		Table 2. Changes in burst frequency during intermittent anoxia in WT and Tac1–/– mice

 
Since brief repetitive hypoxia can induce long-term modification of the respiratory activity both in vivo and in vitro (5, 29, 53), we monitored the changes in the respiratory output 10 and 40 min after the last anoxic episode. We also determined the possible effects of single prolonged anoxia. For these two types of anoxic conditions, the burst amplitude did not differ from control preanoxic values in either mouse strain. Ten minutes after anoxia, the burst frequency also returned to control values in both WT and Tac1–/– preparations. However, 40 min after intermittent anoxia, the frequency was significantly increased again, up to 134.9 ± 24% of control values in WT (n = 7, P < 0.01, t-test with Bonferroni correction), but still no changes (105.4 ± 24%, n = 10) were found in Tac1–/– preparations. The single prolonged anoxia did not induce significant changes in frequencies in WT (114.6 ± 24%, n = 10) or Tac1–/– (119.3 ± 24%, n = 8) preparations (Fig. 5).


Figure 5
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  		Fig. 5. Changes in burst frequency (±SE) 40 min after continuous and intermittent anoxia. *Significant difference between WT and Tac1–/– (one-way ANOVA, P < 0.05), and a significant increase in frequency compared with preanoxia period (P < 0.01, t-test with Bonferroni correction).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
This is the first study demonstrating the involvement of substance P (and/or NKA) in LTF of respiration in newborn mice and that this potentiation also occurs in the brain stem. Moreover, we demonstrate impairment in development of a normal hypoxic respiratory response in transgenic newborn mice. Our data confirm previous observations that, in the early postnatal period, substance P and the activation of NK-1-receptor is important when the organism is challenged by hypoxia (59, 78, 83). It also corroborates previous studies indicating a differential involvement of substance P in the regulation of ventilation frequency and amplitude (8, 52, 68, 82, 83).

Basal conditions.

Deletion of the Tac1 gene did not affect eupnic breathing or respiratory motor output during normoxic conditions at age P2-3. These data are in agreement with other in vitro studies, showing that severe inherited imbalance in substance P-mediated signaling, such as the lack of substance P in PPT-A mutant mice (73) or NK-1 receptors (57, 59), results in apparently normal respiratory activity.

Neonatal apneas (obstructive, mixed, or central) during eupnea may, beyond owing to immaturity of the respiratory network, also be physiological and are vital for maintenance of lung compliance and alveoli recruitment. Our finding that Tac1–/– mice have fewer apneas in vivo at P2-3 during normoxic and hypoxic conditions may reflect a disturbed peripheral input (i.e., from pulmonary stretch receptors or chemoreceptors). Since we found no clear apneic pattern in vitro, we assume that these apneas are of obstructive or mixed type.

Hypercapnia.

Our results show that f increases in response to hypercapnia, both in vivo and in vitro, in both Tac1–/– mice and WT, indicating that substance P is probably not involved in the hypercapnic respiratory response to the same extent as in the response to hypoxia. This is coherent with previous observations (44, 83), and other studies have shown that the serotonergic system is of greater importance for the CO2 balance (64, 72).

Intermittent isocapnic hypoxia and anoxia.

In this study, we could see no clear respiratory response to hypoxia in age group P2-3 in vivo. Although this may be due to methodological aspects, e.g., insensitivity of the plethysmography setup, it could also reflect compensatory mechanisms such as changes in metabolic rate, which is a known hypoxic defence in newborn mammals (40). The hypoxic response was affected in Tac1–/– preparations during anoxic conditions by 1) less amplitude reductions, 2) a shorter recovery period when the burst amplitude was restored, and 3) more frequent PHNA following resumption of oxygenation. Furthermore, unlike in WT preparations, intermittent anoxia did not induce any LTF effect on bursting frequency. The anoxic frequency response in Tac1–/– preparations was similar to WT, whereas PPT-A mutant mice showed an enhanced anoxic response (73). This discrepancy may be explained partly by different genetic background of PPT-A (CD-1) and Tac1–/– (C57BL/6J) mouse strains, and a possible influence of genetic knockout models themselves (6, 84). Thus, in line with previous reports (73, 84), our data suggest that, in Tac1–/– mice, compensatory mechanisms develop during early ontogeny allowing normal respiratory activity under ordinary conditions but not during anoxic stress. The nature of this partial adaptation is not clear, and the compensatory mechanisms might include different respiratory network characteristics and/or substituting neuromodulators. For example, glycine, opioids, adenosine, and serotonin have all been demonstrated to affect PHNA duration (28, 30). Thus one or several of these neuromodulatory systems might exert a compensatory mechanism on the respiratory output in Tac1–/– mice. Although the physiological significance of PHNA is unclear, it may be related to a decreasing metabolism in the period following hypoxia (13) when the respiratory neurons of the ventrolateral medulla recover after reoxygenation (3). This may result in a possible desynchronization within the ventrolateral medullary respiratory network. Since the transgenic mice preparations demonstrate a greater probability of PHNA, this may be a manifestation of a slower recovery and thus a decreased plasticity of the respiratory network and lack of LTF. This also suggests that substance P is involved in facilitating respiratory rhythm generation, as shown previously for the serotonergic system (1, 18, 29). It has also recently been shown that the medullary serotonin system is affected in sudden infant death victims (51). This may be of interest in relation to substance P-ergic neurotransmission, since serotonin and substance P are extensively colocalized in neurons of the ventral medulla (32, 75). Further investigations are needed to understand their functional relationship, also with reference to Rett syndrome (66, 81).

Our observations at P8-10, where VT/body wt increases in response to isocapnic IH in WT but not in transgenic mice, whereas both groups display a similar increase in f, are consistent with previous observations on PPT-A knockout mice (44). Thus Tac1–/– mice display a sustained immaturity of the respiratory network. The increase in VE during posthypoxic periods (Fig. 1F) in WT, previously described in rats (45), probably also reflects a LTF of the respiratory network. The volatility of the central respiratory network is manifested by the prominent posthypoxic frequency decline and concurrent severe depression of VT/body wt below baseline seen in transgenic mice at age P8-10, contrasting with the normal response seen in WT mice (12, 45) (Fig. 1D). This effect has, in other studies, been demonstrated to be due to carotid body activation (2, 5, 37) and to be serotonin dependent (1, 17, 18, 45, 53). We here demonstrate that substance P is also involved in this mechanism and that the posthypoxic frequency decline seems to originate at a central level.

In the analysis of the breathing pattern, we recognized two distinct types of AIB (16) (see Fig. 2, A and B). The pause or apnea following the AIB might be due to augmenting expiratory cells that inhibit other respiratory cells, which might be of importance for the respiratory network to be reorganized (47). We suggest that AIB followed by a pause have the characteristics of a sigh and that AIB without a pause represents another type of sigh activity dominating during hypoxia. The latter may still originate from the same neural network within the ventral respiratory group. AIB is a periodic phenomenon known to prevent atelectasis and to increase lung compliance but may also represent reconfigurative properties of the central respiratory network (20, 34, 41, 71, 74). This is in line with previous data where hypoxia decreased both the inspiratory time in the augmented breath and the refractory time thereafter, indicating a central mechanism of pattern generation (9, 20, 34). Moreover, deficits in sigh activity have been shown in infants to correlate to SIDS (26), and further studies are needed to understand the underlying mechanisms and physiological role of augmented breaths.

In conclusion, we here demonstrate, in vivo and in vitro, that transgenic newborn mice lacking substance P and NKA possess an abnormal respiratory response and breathing pattern during hypoxic, but not hypercapnic, stress. Our results contribute to the understanding of LTF as a consequence of isocapnic IH and indicate that substance P is an important neuromodulator for an adequate respiratory response. Our data also suggest that Tac1 gene deletion results in an impaired plasticity of respiratory control at a central level. A properly functioning substance P-ergic system may therefore be of great importance to avoid the worse clinical outcome in SIDS and the respiratory disturbance seen in patients suffering from Rett syndrome.


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This study was supported by The Swedish Medical Research Council (K2006-27X-20054-01-3), Harald Jeanssons Stiftelse, Stiftelsen Frimurare Barnhuset i Stockholm and Stiftelsen Samariten.


   FOOTNOTES
 

Address for reprint requests and other correspondence: J. Berner, Neonatal Research Unit, Q2:07, Astrid Lindgren Children's Hospital, Karolinska Univ. Hospital, S-171 76 Stockholm, Sweden (e-mail: Jonas.Berner{at}ki.se)

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.


   REFERENCES
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