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Central serotonin (5-HT) neurons modulate many vital brain functions, including respiratory control. Whether breathing depends critically upon 5-HT neurons, or if their influence is excitatory or inhibitory remains controversial. Here we show that neonatal Lmx1bf/f/p mice, which selectively lack serotonin neurons, display frequent and severe apnea lasting as long as 55 seconds. This was associated with a marked decrease in ventilation to less than half of normal. These respiratory abnormalities were most severe during the post-natal period, markedly improving by the time the pups were 2–4 weeks old. Despite the severe breathing dysfunction many of these mice survived, but there was a high perinatal mortality, and those that survived had a decrease in growth rate until the age at which the respiratory defects resolved. Consistent with these in vivo observations, respiratory output was markedly reduced in isolated brainstem-spinal cord preparations from neonatal Lmx1bf/f/p mice, and completely blocked in perfused brain preparations from neonatal rats treated with selective antagonists of 5-HT2A and neurokinin 1 (NK-1) receptors. The ventilatory deficits in neonatal Lmx1bf/f/p mice were reversed in vitro and in vivo with agonists of 5-HT2A and/or NK-1 receptors. These results demonstrate that ventilatory output in the neonatal period is critically dependent on serotonin neurons, which provide excitatory drive to the respiratory network via 5-HT2A and NK-1 receptor activation. These results provide insight into the mechanisms of sudden infant death syndrome (SIDS), which has been associated with abnormalities of 5-HT neurons, and of cardiorespiratory control.
Breathing is a spontaneous, rhythmic motor output critical for maintaining O2, CO2 and pH homeostasis. 5-HT neurons are embedded within the brainstem respiratory network, and are thought to play diverse roles, including providing tonic modulatory input to the respiratory network and as CO2/pH chemoreceptors (Richerson, 2004; Ptak et al., 2009; Corcoran et al., 2009). However, there remains debate about whether 5-HT neurons are required for the generation and/or stability of breathing, and if their influence is excitatory or inhibitory (Hodges and Richerson, 2008).
Data from rhythmically-active slice preparations (Smith et al., 1991) point to a critical role of 5-HT and the co-transmitter substance P (SP). Raphé obscurus 5-HT neurons project directly to the rhythm-generating pre-Bötzinger complex (pre-BötC) and respiratory motor neurons (Ptak et al., 2009). Within slices, respiratory motor output is blocked with antagonists of 5-HT and/or NK-1 receptors (Pena and Ramirez, 2002; Telgkamp et al., 2002; Gunther et al., 2006; Ptak et al., 2009), and stimulation of the raphé obscurus increases respiratory burst frequency (Al-Zubaidy et al., 1996; Ptak et al., 2009).
In contrast, the role of 5-HT neurons in the intact nervous system is less clear. For example, eupneic respiratory output in juvenile in situ perfused brain preparations is not prevented by antagonists of 5-HT receptors (Toppin et al., 2007), and 5-HT receptor antagonists do not inhibit breathing in adults in vivo (Ling et al., 2001; McGuire et al., 2004). Blocking 5-HT synthesis in vivo can cause an increase in ventilation, suggesting 5-HT neurons may inhibit breathing (Olson, Jr. et al., 1979). A possible explanation for these contradictory results is that it is necessary to block the effects of co-released neuropeptides, in addition to 5-HT, to significantly reduce excitability of the respiratory network. Consistent with this, a combination of 5-HT and NK-1 receptor antagonists eliminates respiratory output in juvenile in situ perfused brain preparations (Ptak et al., 2009). Similar experiments have not yet been performed in vivo.
Lesions of 5-HT neurons with toxins such as 5,7-dihydroxytryptamine (Mueller et al., 1984; Cummings et al., 2009), or anti-SERT-saporin (Nattie et al., 2004) have inconsistent effects on baseline ventilation, possibly due to a variable percentage of remaining 5-HT neurons. Pet-1 knockout mice have only 30% of the normal number of 5-HT neurons, but display only mildly disrupted breathing as neonates (Erickson et al., 2007). Knockout of the transcription factor Lmx1b in Pet-1-expressing neurons (Lmx1bf/f/p) specifically eliminates all 5-HT neurons (Zhao et al., 2006). As adults, these mice have a decreased ventilatory response to hypercapnia and reduced baseline respiratory frequency, but only a small and inconsistent reduction in baseline ventilation (Hodges et al., 2008; Hodges and Richerson, 2008b). Taken together, these data have been interpreted by some as indicating that there is little if any stimulation of baseline breathing by 5-HT neurons in vivo.
Here we study neonatal Lmx1bf/f/p mice and demonstrate a critical, excitatory influence of 5-HT neurons on breathing in vivo and in vitro mediated by 5-HT2A and NK-1 receptor agonists. This dependence on 5-HT neurons is lost as these mice develop into adults.
All procedures and protocols were approved by the Yale Animal Care and Use Committee, or by the NINDS Animal Care and Use Committee prior to study.
Generation of Lmx1bf/f/p mice has been previously described (Zhao et al., 2006). Females that were homozygous for floxed Lmx1b (Lmx1bf/f) were bred with male mice that were homozygous for floxed Lmx1b and hemizygous for ePet1-Cre (Lmx1bflox/flox;ePet-Cre/+) to generate progeny of these two genotypes in a 1:1 ratio. Lmx1bflox/flox;ePet-Cre/+ mice, also referred to as Lmx1bf/f/p mice, fail to develop central 5-HT neurons, whereas Lmx1bf/f mice are phenotypically normal and have a normal complement of 5-HT neurons (referred to here as WT). Tail biopsy samples were obtained on or before the fourth postnatal day of life for genotyping, using procedures previously described (Zhao et al., 2006).
Ventilation was measured in vivo using whole body plethysmography with methods similar to those described previously (Hodges et al., 2008; Hodges and Richerson, 2008b). Two complementary approaches were used: 1) Flow-through, and 2) Stop-flow. Both approaches allow ventilatory measurements from unrestrained mice, so that resting breathing was unperturbed, allowing accurate measurements of frequency and apneas. Stop-flow plethysmography has the advantage that it allows low-noise recordings of small amplitude breaths in very young neonatal mice. This approach was used for all of the apnea studies (from P2 to P20) so that small breaths were not missed and mistakenly categorized as apnea. This approach does not allow calculation of actual tidal volumes, so tidal volume and ventilation were expressed relative to WT values. Flow-through plethysmography was used to study a separate subgroup of mice aged P2-P28, including some that were older than P12 in which temperature probes were implanted to allow tidal volumes and minute ventilation to be calculated. This approach has the added advantage of allowing measurements of oxygen consumption, thus permitting normalization of ventilation to metabolic rate.
Baseline ventilation was measured in two sets of WT and Lmx1bf/f/p mice at ages from P2 to P28 in plethysmographs of different volumes (9 ml, 15 ml, and 28 ml), to maximize the ventilation signal and allow for growth. As described previously (Hodges et al., 2008; Hodges and Richerson, 2008b), each chamber had ports for gas inflow and outflow, a pressure transducer, and a relative humidity and ambient temperature sensor. During flow-through experiments tidal volumes were calibrated using a small animal ventilator (Minivent, Harvard Apparatus, Holliston, MA) by injecting 0.1 to 0.3 ml of air at a rate of 1.5 Hz. Oxygen consumption (VO2) was calculated by subtracting the outflow fraction of O2 from the inflow fraction of O2, and multiplying the difference by the chamber flow rate measured using a gas flowmeter (PV500CCMVADA-CP, Cole-Parmer, Vernon Hills, IL). Animal temperatures were measured before and after periods of study with j-type thermocouple probes for rectal temperature (BAT-12; Physitemp Instruments, Inc., Clifton, NJ) or with telemetric temperature probes (Bio Medic Systems, Inc., Seaford, DE), implanted as described previously (Hodges et al., 2008). Baseline ventilation was recorded at ambient temperatures of 24°C (room temperature), or when the plethysmography chamber was warmed to either 27°C or 30°C using a temperature controller (TCAT-2AC, Physitemp, Instruments, Inc., Clifton, NJ) and an incandescent lamp.
Data was continuously acquired with a custom-written data acquisition program written in Matlab (The MathWorks, Natick, MA) after digitization using an analog-to-digital converter (PCI-6221, National Instruments, Austin, TX). Pressure transducer output was digitized at 100 Hz, and ambient temperature, relative humidity, and O2 concentrations were digitized at 0.1 Hz. Breathing-induced pressure oscillations were analyzed offline using a custom-written Matlab program that detected peaks and troughs with manual confirmation by the investigator.
En bloc experiments were performed and analyzed by an individual blind to animal genotype, using methods similar to those previously described (Smith et al., 1990). Two day-old WT and Lmx1bf/f/p mice were deeply anesthetized with isoflurane, decapitated, and the brainstem-spinal cord removed and transected at the level of the rostral pons in ice-chilled artificial cerebrospinal fluid (aCSF) containing (in mM): NaCl 129; NaHCO3 21; KCl 3; CaCl2 1.5; MgSO4 1.0; NaH2PO4 0.58; dextrose 30; pH 7.35–7.4 after bubbling with 95% O2/5% CO2. The preparation was transferred to a recording chamber and superfused with aCSF of the same composition, except that KCl was increased to 4.5 mM. DOI hydrochloride (10 μM; Sigma-Aldrich, St. Louis MO) and substance P acetate salt hydrate (1 μM) were dissolved in aCSF and supplied via the superfusate. Bath temperature was maintained at 27 – 27.5°C with a TC-324B temperature controller (Warner Instruments, Hamden, CT).
Hypoglossal (XII) and cervical (C1–4) nerve signals were measured via suction electrodes, amplified (×10,000) and band-pass filtered (0.3 – 1 kHz) with a Grass LP511 AC (Astro-med, Inc, West Warwick, RI) and DX4-400 differential amplifier (Dagan Corp., Minneapolis, MN), and 60 Hz noise reduced with a Hum Bug noise eliminator (Quest Scientific, North Vancouver, BC). The signal was digitized at 100 Hz, and acquired and analyzed offline using custom-written software in Matlab. Nerve activity was integrated, and respiratory bursts (onset, peak and end) detected using a user-defined threshold. The peak of integrated bursts was compared to a baseline value (300 ms before burst onset) to obtain amplitude.
Experiments were performed with in situ arterially perfused brainstem-spinal cord preparations from 6–8 day old neonatal rats, which generate patterns of cranial and spinal motoneuron activity similar to those in vivo (Paton, 1996; Dutschmann et al., 2000; Smith et al., 2007). Procedures for setting up the in situ arterially perfused brainstem-spinal cord preparations of neonatal rats (Dutschmann et al., 2000) were as described previously in detail for juvenile rats (Paton, 1996; Smith et al., 2007). In brief, pre-heparinized (1000 units i.p.) P6–P8 male Sprague-Dawley rats were anaesthetized deeply with isoflurane until loss of the paw withdrawal reflex. Rats were bisected sub-diaphragmatically, the head and thorax was immersed in ice-chilled carbogenated Ringer’s solution, and the brain decerebrated pre-collicularly. Thoracic phrenic (PN) and hypoglossal (XII) nerves were cut distally. Preparations were transferred to a recording chamber. A double lumen cannula (modified DLR-4, Braintree Scientific, MA, USA) was inserted retrogradely into the descending aorta for perfusion. Perfusion solution was supplied via a peristaltic roller pump (Watson Marlow 505D) and consisted of (in mM): NaCl 125; NaHCO3 24; KCl 3; CaCl2 2.5; MgSO4 1.25; KH2PO4 1.25; dextrose 10; pH 7.35–7.4 after bubbling with 95% O2/5% CO2 at 32°C. Osmolality was 290 ± 5 mOsm·kg H2O−1. Ficoll 70 (1.25%) was added as an oncotic agent. The second lumen of the cannula was used to monitor aortic perfusion pressure. Vasopressin (200–400 pM as required) was added to the perfusate to raise perfusion pressure to between 70–90 mmHg (Pickering and Paton, 2006). Vecuronium bromide (4 μg·ml−1; Organon Teknica, Cambridge, UK) was added to the perfusion solution to block neuromuscular transmission. Methysergide maleate (Sigma-Aldrich, St. Louis MO; dissolved in 10% DMSO), SR 140333 (a gift from Sanofi-Aventis, Bridgewater, NJ; dissolved in 7% ethanol), or MDL 11,939 (Tocris Bioscience, Bristol, UK; dissolved in 100% ethanol) were diluted in the perfusate solution.
Simultaneous PN and XII nerve recordings were obtained with bipolar suction electrodes in situ. All nerve signals were amplified (x5,000 – 10,000) and band-pass filtered (0.3 – 1 kHz) with a Cyberamp 360 amplifier (Molecular Devices, Sunnyvale, CA). Raw recordings were rectified and smoothed by analog or digital integration (30 – 100 ms time constant) and simultaneously acquired digitally (10 kHz sampling rates) using a PowerLab A/D converter and Chart v5.0 software (AD Instruments, Inc., Colorado Springs, CO). The parameters describing network population activity (XII n. and PN inspiratory interburst interval/frequency and burst amplitude) were calculated offline using Chart v5.0 and Igor Pro (Wavemetrics, Portland, OR) software with custom automated analysis procedures, and hand-checked for accuracy.
Statistical significance was tested using χ2 analysis, and group means were compared using a two-sample T-test assuming unequal variances when appropriate (MS Excel). One-way or two-way ANOVA were used for comparing effects of genotype, age, temperature, or drug and their interactions, with either Newman-Keul’s (Prism 4, GraphPad Software), or Tukey (Systat 11, Systat Software, Inc., Chicago, Il.) post-hoc analyses for multiple comparisons. The threshold for significance was set to P < 0.05. All data are presented as mean ± SEM.
In contrast to adults (Hodges et al., 2008), neonatal Lmx1bf/f/p mice had severely disrupted baseline breathing (Figs. 1A,B). Lmx1bf/f/p pups had short trains of low amplitude, low frequency breaths interrupted by spontaneous and severe apnea, lasting as long as 55 seconds in extreme cases (Fig. 1A). Wild type (WT) pups also occasionally had apnea (defined as an interbreath interval longer than 1 second) during the neonatal period. However, apneas in Lmx1bf/f/p pups were much more frequent, longer, and persisted to a later postnatal age (Figs. 1B–D). At postnatal day 2 (P2), Lmx1bf/f/p pups studied at warm ambient temperature (30°C) spent 39.2% of the time apneic (calculated as apnea duration * frequency/total study time), compared to 5.8% for WT pups (Fig. 1C). Similar results were obtained at an ambient temperature of 24°C, including the percentage of time apneic (Fig. 1C), apnea frequency (Fig. 1C (inset)) and apnea duration (data not shown).
There was even greater disparity between WT and Lmx1bf/f/p pups when comparing prolonged apneas (>5 seconds). Combining data obtained at ambient temperatures of 24°C and 30°C, 100% of Lmx1bf/f/p pups exhibited at least one prolonged apnea when studied for a 5-minute period at P2, compared to only 20% of WT pups (Fig. 1D). At this age, we observed 1.3 ± 1.3 prolonged apneas/hour in WT pups, with a mean duration (5.8 ± 0.1 s) slightly greater than the defined threshold of 5 s, compared to 52.9 ± 12.5 prolonged apneas/hour in Lmx1bf/f/p pups (Fig. 1E) with a mean duration of 9.6 ± 0.7 s.
Resting ventilation was markedly lower in neonatal Lmx1bf/f/p mice compared to WT mice when normalized either to weight (and expressed as a percentage of WT ventilation) or oxygen consumption (Figs. 2A–C). This was a consistent finding in multiple datasets collected using different approaches, including flow-through and stop-flow plethysmography (see Methods), or when measurements were made at different ambient temperatures (24, 27 or 30°C). The reduced baseline ventilation was due to both a smaller breath amplitude (Fig. 2D) and lower breathing frequency (Fig. 2E). Breathing was also much more irregular in Lmx1bf/f/p mice, with the coefficient of variation of the interbreath interval (CV IBI) much greater than WT mice (Fig. 2F). Animal temperatures were slightly lower in Lmx1bf/f/p mice at P12, P20 and P28 (Fig. 2G), consistent with previous observations of a thermoregulatory deficit (Hodges et al., 2008; Hodges and Richerson, 2008b). However, the small differences could not explain the marked difference in ventilation between genotypes, and animal temperatures were equal to WT mice at all other ages studied. Most Lmx1bf/f/p mice spent very little time breathing with a normal regular (eupneic) pattern, instead often moving air primarily via an erratic pattern (Figs. 1A & 6A) or during vocalization (Fig. 1B). At P4, vocalization amplitude (normalized to weight) in Lmx1bf/f/p (0.0682 ± 0.005 a.u.) mice was not different compared to WT (0.0706 ± 0.005 a.u.; P = 0.74) mice, indicating that the neuromuscular system of Lmx1bf/f/p mice was capable of generating large tidal volumes and that our methods were capable of accurately measuring them. Thus, the severe ventilatory phenotype in Lmx1bf/f/p mice demonstrates a critical contribution of 5-HT neurons to normal eupneic respiratory output in early development in vivo.
In parallel with the severe respiratory phenotype, many Lmx1bf/f/p pups died during the neonatal period. Tail tissue was collected on or before P4 for genotyping from 419 WT and Lmx1bf/f/p mice reared at an ambient temperature of 24°C. The expected ratio of WT to Lmx1bf/f/p mice based on a Mendelian pattern of inheritance was 1:1, whereas the actual ratio was 234 WT to 185 Lmx1bf/f/p mice (χ2; P = 0.017), giving an estimated excess mortality rate of 21% in Lmx1bf/f/p mice during the neonatal period.
Some Lmx1bf/f/p mice with extremely long apneas as neonates died before reaching adulthood. For example, the P4 Lmx1bf/f/p mouse shown in Fig. 1A died at P9, and another Lmx1bf/f/p mouse with severe (>55 seconds) apnea at P6 died at P14. However, the majority of Lmx1bf/f/p mice survived beyond P28 despite the prevalence of prolonged apnea in all Lmx1bf/f/p mice at P2.
In the Lmx1bf/f/p mice that survived the neonatal period, there was marked improvement in all apnea measures with increasing age (Fig. 1B). The percentage of time apneic, apnea frequency, apnea duration and percentage of animals exhibiting apnea steadily declined with age in Lmx1bf/f/p pups, completely disappearing around P15–P20 (Fig. 1C). In parallel with the reduction in apnea, there was an increase in baseline ventilation, breath amplitude and frequency in Lmx1bf/f/p mice. In fact, at P12 ventilation, breath amplitude and frequency of Lmx1bf/f/p mice studied at an ambient temperature near thermoneutral (30°C) was equal to that of WT mice (Fig. 2C–E). At lower ambient temperature (24°C or 27°C), the deficit in minute ventilation and reduced VE/VO2 persisted to at least P28 before both measures normalized in adults (Figs. 2A–C). Breathing frequency was lower in Lmx1bf/f/p mice throughout development measured at 24°C or 27°C (Fig. 2E; data not shown), and as shown previously remained lower in adult Lmx1bf/f/p mice (Hodges et al., 2008; Hodges and Richerson, 2008b).
In parallel with abnormal respiratory control, there was a lower growth rate in Lmx1bf/f/p mice during early development that improved with age. Birth weights were equal for WT and Lmx1bf/f/p pups (Fig. 3A (inset)), but beginning at P1 weights were lower in Lmx1bf/f/p pups (Fig. 3A) due to a lower growth rate (Fig. 3B). Coincident with the time apnea resolved near P15, there was an increase in growth rate in Lmx1bf/f/p mice (Fig. 3B), leading to normalization of body weight by P55 (Fig. 3A). These results suggest that the initial lag in weight gain was due to growth retardation from hypoxia (Mortola et al., 1990), which presumably resulted from severe apnea.
The apnea and hypoventilation in Lmx1bf/f/p mice could possibly be due to reduced excitatory serotonergic input to the respiratory network (Pena and Ramirez, 2002; Telgkamp et al., 2002; Ptak et al., 2009), abnormal embryonic development of the respiratory network due to the absence of 5-HT (Bou-Flores et al., 2000), or an indirect influence from abnormalities outside the CNS. To distinguish between these possibilities, we performed recordings from isolated brainstem-spinal cord (en bloc) preparations from P2 WT and Lmx1bf/f/p mice (Fig. 4A). Spontaneous, rhythmic respiratory bursting was present in the hypoglossal (XII) and cervical (C1–4) nerves of all WT mice (Figs. 4A,B). In contrast, respiratory output was infrequent (Fig. 4B) and irregular in Lmx1bf/f/p mice. The addition of the 5-HT2A agonist 1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane (DOI) to the superfusate had little effect on burst frequency in WT mice, but increased burst frequency in Lmx1bf/f/p preparations to that of WT mice (Fig. 4C). There were no differences in burst duration or amplitude between genotypes under control conditions or during DOI treatment (Figs. 4D,E). The application of SP (which is co-localized in subpopulations of 5-HT neurons in mice (Holtman, Jr. et al., 1984; Hokfelt et al., 2000; Ptak et al., 2009) in combination with DOI significantly increased respiratory burst frequency in both WT and Lmx1bf/f/p preparations, with an initial large transient, and subsequent smaller sustained increase in frequency (Fig. 4C). In a different set of Lmx1bf/f/p en bloc preparations we also found that substance P alone (Figs. 4F,G) or 5-HT (data not shown) also increased burst frequency similar to DOI, whereas increasing [K+] to 6 mM, applied to increase respiratory network excitability, caused an increase in frequency that was only transient (Figs. 4H,I). These data show that the reduced respiratory output in Lmx1bf/f/p mice is due to a lack of neuromodulatory drive to an intact and functional respiratory network, and demonstrate an important role for endogenous central 5-HT and NK-1 receptor activation during early development.
Endogenously released 5-HT and SP are well known to modulate inspiratory network activity in vitro (Telgkamp et al., 2002; Pena and Ramirez, 2002; Ptak et al., 2009), but it has been controversial whether breathing critically depends upon these neurotransmitters in the intact CNS (Al-Zubaidy et al., 1996; Toppin et al., 2007). We used P6-P8 rat pups for perfused neonatal brainstem-spinal cord (in situ) preparations, which offer the advantage that they have a more intact nervous system than en bloc in vitro preparations (Richerson and Getting, 1990; Paton, 1996) and allow application of drugs that can cause confounding (e.g. peripheral) effects in vivo. Receptor antagonists were delivered intravascularly via the perfusate to obtain detailed dose-response relationships. 5-HT receptors were blocked with methysergide, a 5-HT1/2 receptor competitive antagonist that is also a weak partial agonist at 5-HT1A/B/D receptors, or MDL 11,939 (MDL), a highly selective 5-HT2A receptor antagonist. NK-1 receptors were blocked by SR 140333 (SR). Methysergide progressively reduced the amplitude (P < 0.001) and frequency (P < 0.001) of inspiratory motor output recorded from phrenic (PN) and XII nerves over the concentration range tested (5–20 μM, n = 3), ultimately eliminating both motor outputs completely in all preparations (data not shown). Similarly, increasing concentrations of MDL also progressively reduced inspiratory burst frequency and amplitude in all preparations (n = 4) over the concentration range tested (5–40 μM, Figs. 5A–D), and completely eliminated motor output at the highest MDL concentration (40 μM). SR also progressively and significantly reduced inspiratory PN and XII burst frequency and amplitude, eliminating inspiratory motor output in 3 of 4 preparations at 40 μM and in all preparations at 50 μM (Figs. 5B–D). Thus, as recently shown in mature rats using a perfused brain preparation (Ptak et al., 2009), and consistent with the data presented here from neonatal mice lacking 5-HT neurons, neural respiratory output in immature rats is highly dependent upon endogenous activation of 5-HT2A and NK-1 receptors.
Based on these data, we hypothesized that 5-HT2A receptor activation in vivo may improve the frequent and severe apnea observed in Lmx1bf/f/p mice during early postnatal development. Subcutaneous injections of DOI (0.1 μg/gm) in Lmx1bf/f/p pups at the age where apnea is most severe caused significant improvement in apnea indices and resting ventilation (Fig. 6A). One hour after injection, ventilation was 3.1-fold greater, and the percentage of time apneic decreased from 26 ± 5% to 10 ± 3% (Fig. 6B), a level similar to untreated WT pups at P2 (6 ± 2%). The improvement in ventilation and apnea was sustained for more than 4 hours (Fig. 6B). Other measures of ventilatory function were also improved with DOI treatment. Apnea duration (2.3 ± 0.4 vs. 1.5 ± 0.1 s; P = 0.038) and the coefficient of variation of the interbreath interval (0.90 ± 0.21 vs. 0.53 ± 0.06; P = 0.041) both decreased, whereas breathing frequency (68.2 ± 7.0 vs. 127.6 ± 7.2 breaths/min; P < 0.001) and breath amplitude (0.0075 ± 0.0007 vs. 0.0124 ± 0.0007 a.u.; P = 0.016) increased 2 hours after DOI injection. Although many aspects of ventilatory dysfunction were improved with systemic DOI treatment, we also observed side effects including tremor and myoclonus. These data suggest that 5-HT2A receptor activation can markedly improve ventilation and apnea in neonatal mice with severe 5-HT system dysfunction, but also suggest an alternative drug or more targeted application may be necessary to avoid side effects.
This study demonstrates that 5-HT neurons are required for normal respiratory output in vivo during early development. Neonatal mice devoid of central 5-HT neurons displayed frequent and severe apnea, hypoventilation, excess mortality at the time breathing abnormalities were the most severe (<P4), and growth retardation. The respiratory dysfunction in Lmx1bf/f/p mice was also apparent in vitro, where it was normalized with 5-HT2A and/or NK-1 receptor activation. Consistent with these results, blockade of endogenous 5-HT2A and/or NK-1 receptor activation eliminated respiratory motor output in neonatal in situ perfused preparations from rats. Importantly, the ventilatory dysfunction and growth retardation in Lmx1bf/f/p mice that survived the neonatal period normalized with increasing age, suggesting that either: 1) the contributions of 5-HT neurons to baseline ventilation decreases with age, or 2) long-term compensation within the respiratory network acts to mitigate effects of 5-HT neuron loss. These data provide clear evidence that the primary influence of 5-HT neurons in respiratory control is excitatory, and that they are critical for normal ventilation during early development.
The breathing dysfunction and reduced respiratory output in vivo and in vitro in Lmx1bf/f/p mice could theoretically result from either improper network formation due to loss of the trophic effects of 5-HT (Bou-Flores et al., 2000), and/or a lack of excitatory, neuromodulatory drive (Hodges and Richerson, 2008) due to severe depletion of central 5-HT (Zhao et al., 2006), and the co-transmitters SP and TRH. However, the data point to the latter possibility since the respiratory output in en bloc preparations from Lmx1bf/f/p mice was normalized with 5-HT, DOI, and substance P. In addition, the improvement of apnea indices and the appearance of rhythmic, eupneic ventilation with a 5-HT2A agonist in vivo suggests that Lmx1bf/f/p mice have properly developed a functional respiratory network that simply lacks sufficient excitatory neuromodulatory input. The specific site(s) of action for these neuromodulatory effects are unclear, but likely include the pre-Bötzinger complex and respiratory motor neurons based on previous observations in rhythmic slice preparations (Al-Zubaidy et al., 1996; Pena and Ramirez, 2002; Ptak et al., 2009). The elimination of respiratory network output in situ with selective antagonists for 5-HT2A and NK-1 receptors in neonatal and juvenile preparations (Ptak et al., 2009) further supports this conclusion.
There has been considerable controversy regarding whether 5-HT neurons provide any significant input to the respiratory network in vivo, or whether it is excitatory. Here we show there is a critical dependence of normal breathing on 5-HT neurons during early development. The role and relative importance of 5-HT neurons in adults in vivo remains less clear. Adult Lmx1bf/f/p mice exhibit relatively normal, stable eupneic ventilation despite the near-complete lack of 5-HT neurons (Hodges et al., 2008). It is certainly possible that there is only a transient dependence of the neural respiratory networks on 5-HT and/or substance P, and that these neurons are superfluous or redundant in adults. However, there are alternative interpretations of the existing data, and it remains possible that 5-HT neurons provide essential excitatory input throughout life (Ptak et al., 2009). For example, there is a marked capacity for plasticity in the developing brain, and the normal baseline breathing observed in adult Lmx1bf/f/p mice may be a result of compensatory changes that lead to replacement of 5-HT input by an alternative source of excitatory drive. The source of such drive is unknown, but may include increased reliance upon peripheral chemoreceptors, or other central sources, including medullary (Zanella et al., 2006) or pontine (Hilaire et al., 2004) catecholaminergic neurons. Alternatively, other modes of plasticity could contribute to the ventilatory compensation, such as changes in intrinsic properties of neurons within the respiratory network, as might be expected if the principles of homeostatic plasticity apply to brainstem circuits (Turrigiano and Nelson, 2004).
Previous experiments addressing the issue of the importance of 5-HT neurons for normal respiratory output in adults in vivo are subject to a variety of confounding factors. For example, systemic treatment with 5-HT receptor antagonists does not consistently decrease baseline breathing in vivo or in situ (McGuire et al., 2004; Toppin et al., 2007). Agents that block synthesis of 5-HT cause an increase in breathing (Olson, Jr. et al., 1979). However, these approaches would have no effect on SP or TRH signaling, both of which are strongly stimulatory to breathing (Cream et al., 1999; Telgkamp et al., 2002; Richerson, 2004). Thus, endogenous release of SP and TRH alone may be sufficient to maintain eupnea after blocking 5-HT receptors (Ptak et al., 2009), and blocking 5-HT synthesis could actually increase ventilation due to loss of autoinhibition, which could cause greater release of SP and TRH. Lesioning or silencing 5-HT neurons in some cases decreases baseline breathing (Mueller et al., 1984), but in other cases has no effect (Nattie et al., 2004; Cummings et al., 2008). However, it is necessary to use focal injections for these approaches, and it is difficult to influence all 5-HT neurons since they are so widely distributed throughout the medulla. In addition, symptoms do not usually develop from loss of monoaminergic neurons until a large percentage (>70–80%) of them are lost (Blier, 2008). Thus, further work will be needed to define the relative importance of 5-HT neurons for eupnea in adult mammals in vivo.
The work presented here does not address the related question of whether 5-HT neurons are central CO2 chemoreceptors. Significantly more work will be needed to define whether there are changes in the acute responses to hypercapnia or hypoxia in neonatal Lmx1bf/f/p mice. Nonetheless, a prerequisite for acidosis-stimulated 5-HT neurons to be important as central respiratory CO2 chemoreceptors is that they must provide excitatory input to the respiratory network. The work presented her clearly demonstrates that this is the case, supporting the previous conclusion (Richerson, 2004; Corcoran et al., 2009) that the increase in firing rate of a subset of 5-HT neurons that occurs in response to acidosis both in vitro (Wang et al., 1998; Wang et al., 2001) and in vivo (Larnicol et al., 1994; Veasey et al., 1995; Haxhiu et al., 1996; Teppema et al., 1997; Haxhiu et al., 2001; Pete et al., 2002; Okada et al., 2002; Johnson et al., 2005; Kanamaru and Homma, 2007) has the capacity to cause an increase in respiratory output.
Understanding the nature of the role of 5-HT neurons in respiratory control during development has become more important in light of recent findings of multiple 5-HT system abnormalities in sudden infant death syndrome (SIDS) (Paterson et al., 2006; Kinney et al., 2008). Limited physiologic data in infants show episodes of apnea and bradycardia prior to a SIDS death (Meny et al., 1994; Poets et al., 1999), consistent with the long-standing hypothesis that SIDS is related to a brainstem abnormality in the neuroregulation of cardiorespiratory control (Hunt and Brouillette, 1987). It is not yet known whether the anatomical defects identified in SIDS cases are associated with an increase or decrease in function of the 5-HT system (Paterson et al., 2006; Kinney et al., 2008), but better defining the roles for 5-HT neurons in control of breathing during development using transgenic animal models (Erickson et al., 2007; Audero et al., 2008; Nattie, 2009; Alenina et al., 2009) may provide insight into how 5-HT neuron defects could lead to pathophysiological changes that cause SIDS.
This work was supported by the Parker B. Francis Foundation (M.R.H), the NICHD, Bumpus Foundation and VAMC (G.B.R), and the Intramural Research Program of NIH, NINDS (J.A. & J.C.S.).
There are no conflicts of interest.