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. 2014 Nov 3;593(Pt 1):305–319. doi: 10.1113/jphysiol.2014.279794

Prostaglandin E2 differentially modulates the central control of eupnoea, sighs and gasping in mice

Henner Koch 1,2,3, Cali Caughie 1, Frank P Elsen 1, Atsushi Doi 1,2, Alfredo J Garcia 1, Sebastien Zanella 1,2, Jan-Marino Ramirez 1,2,✉
PMCID: PMC4293069  PMID: 25556802

Abstract

Prostaglandins are important regulators of autonomic functions in the mammalian organism. Here we demonstrate in vivo that prostaglandin E2 (PGE2) can differentially increase the frequency of eupnoea (normal breathing) and sighs (augmented breaths) when injected into the preBötzinger complex (preBötC), a medullary area that is critical for breathing. Low concentrations of PGE2 (100–300 nm) increased the sigh frequency, while higher concentrations (1–2 μm) were required to increase the eupnoeic frequency. The concentration-dependent effects were similarly observed in the isolated preBötC. This in vitro preparation also revealed that riluzole, a blocker of the persistent sodium current (INap), abolished the modulatory effect on sighs, while flufenamic acid, an antagonist for the calcium-activated non-selective cation conductance (ICAN) abolished the effect of PGE2 on fictive eupnoea at higher concentrations. At the cellular level PGE2 significantly increased the amplitude and frequency of intrinsic bursting in inspiratory neurons. By contrast PGE2 affected neither excitatory nor inhibitory synaptic transmission. We conclude that PGE2 differentially modulates sigh, gasping and eupnoeic activity by differentially increasing INap and ICAN currents in preBötC neurons.

Key points.

  • Prostaglandin E2 (PGE2) augments distinct inspiratory motor patterns, generated within the preBötzinger complex (preBötC), in a dose-dependent way. The frequency of sighs and gasping are stimulated at low concentrations, while the frequency of eupnoea increases only at high concentrations.

  • We used in vivo microinjections into the preBötC and in vitro isolated brainstem slice preparations to investigate the dose-dependent effects of PGE2 on the preBötC activity.

  • Synaptic measurements in whole cell voltage clamp recordings of inspiratory neurons revealed no changes in inhibitory or excitatory synaptic transmission in response to PGE2 exposure.

  • In current clamp recordings obtained from inspiratory neurons of the preBötC, we found an increase in the frequency and amplitude of bursting activity in neurons with intrinsic bursting properties after exposure to PGE2.

  • Riluzole, a blocker of the persistent sodium current, abolished the effect of PGE2 on sigh activity, while flufenamic acid, a blocker of the calcium-activated non-selective cation conductance, abolished the effect on eupnoeic activity caused by PGE2.

Introduction

Breathing has to adapt continuously and differentially to changes in the external and internal environment. At the centre of this adaptation lie neuronal networks that provide the flexibility to respond to ongoing changes. The preBötzinger complex (preBötC; Smith et al. 1991; Schwarzacher et al. 2011), a region that is essential for the generation of breathing (Ramirez et al. 1998; Wenninger et al. 2004; Tan et al. 2008), has been implicated in the modulatory response of breathing. This region, located within the ventrolateral medulla, is the target of several neuromodulatory systems (Doi & Ramirez, ,; Koch et al. 2011; Ramirez et al. 2012). In addition to the classic neuromodulators such as noradrenaline (norepinephrine) (Ellenberger et al. 1990; Viemari & Ramirez, 2006; Zanella et al. 2014), substance P (Gray et al. 1999; Peña & Ramirez, 2004); acetylcholine (Shao et al. 2005) and serotonin (Peña & Ramirez, 2002; Ptak et al. 2009), this network is also regulated by molecules that can be activated through non-neuronal pathways. Some of these molecules are part of the inflammatory pathway and include prostaglandins, which are produced by cyclooxygenase-2 (COX-2) enzymes. Under physiological conditions, these inducible enzymes are expressed at basal levels in the brain. But a number of stimuli, including peripheral infections, pain, traumatic injury, hypoxia and hyperoxia, raise COX-2 expression and subsequently increase protein levels of prostaglandin E2 (Yamagata et al. 1993; Perez-Polo et al. 2011). PGE2, the major reaction product of the COX-2 enzymes, has been implicated in directly modulating the neuronal activity involved in several regulatory systems, including the regulation of pain (Ahmadi et al. 2002), sleep and wakefulness (Takemiya, 2011), induction of fever (Scammell et al. 1996; Lazarus et al. 2007), synaptic plasticity and transmission (Akaneya & Tsumoto, 2006; Koch et al. 2010) and in the control of autonomic functions including respiration (Hofstetter et al. 2007).

Here we characterized the effects of PGE2 on the preBötC using in vitro slice preparations and in vivo preparations from freely breathing animals. Our data demonstrate that low concentrations (<300 nm) of PGE2 injected into the preBötC in vivo increased sigh frequency, but had no effect on normal breathing (eupnoea). Higher concentrations (1–2 μm) of PGE2 were required to also increase eupnoeic frequency. This response was mimicked in the preBötC isolated in an in vitro brainstem slice preparation. Thus our study contributes to the notion that PGE2 is an important modulator of respiratory activity. A comparison with existing studies (Ballanyi et al. 1997; Hofstetter et al. 2007) suggests that prostaglandins exert a variety of diverse effects that may result in adaptive and maladaptive responses of the respiratory network in health and disease.

Methods

All animal experiments were performed using protocols approved by the Institutional Animal Care and Use Committee at Seattle Children’s Research Institute. Mice were maintained with rodent diet and water available ad libitum in a vivarium with a 12 h light/dark cycle at 22°C.

In vivo recordings and microinjection into the preBötC

A total of six CD1 mice (postnatal days (P) 9–16) were anaesthetized with urethane (1.5 g kg−1) by an intraperitoneal injection for in vivo measurements. At the end of the experiments animals were killed by transcardiac perfusion under terminal urethane anaesthesia. Mice, of either sex, were placed in a supine position, and the head was fixed with a stereotaxic apparatus. The neck of the mouse was opened from the ventral side, the trachea was cut, and plastic Y-shaped tubing for supplying oxygen was inserted into the proximal end of the trachea (cannulation). The bone of the skull covering the ventral brainstem was partially removed with small scissors and forceps. The dura and arachnoid membrane were removed to expose the ventral medulla. The surface of the ventral medulla was continuously perfused with 95% O2–5% CO2-equilibrated artificial cerebrospinal fluid (aCSF) solution at 30° ± 0.5°C. In all cases, 100% oxygen was supplied through cannulation without artificial ventilation. The core body temperature during in vivo experiments was measured routinely in our laboratory and was stable at 36° ± 1°C.

Electromyography recordings (EMG) of the intercostal muscles were recorded with a Teflon-covered Ag bipolar electrode. The skin over the abdominal and intercostal area on the right side was partially removed, and the bipolar electrode was placed on the surface of the intercostal muscles. Signals were AC amplified and bandpass filtered (8 Hz to 3 kHz).

Microsyringes (Hamilton microsyringe no. 80330, Hamilton, Reno, NV, USA) with 33 gauge needles containing PGE2 were positioned with a micromanipulator for subsequent microinjections (KITE, World Precision Instruments, Sarasota, FL, USA). The needles of the microsyringes were inserted into the right preBötC from the ventral side. While constantly measuring intercostal EMG activity, PGE2 was injected into the right preBötC (0.3 μl min−1). We did not attempt any bilateral injections to limit the damage to the preBötC, which would have compromised respiratory rhythm generation. As previously described and anatomically characterized, injections in the preBötC are routinely traced and localized to an area encompassing the preBötzinger area in close proximity to the nucleus ambiguus (Zanella et al. 2014). As also demonstrated in previous studies, the injections typically encompass an area that slightly exceeds the preBötzinger area, and the reader is referred to the study by Zanella et al. (2014) for further details.

The transverse slice preparation

Brainstem transverse slice preparations from CD1 (n = 65) mice of either sex (P0–15, Charles River Laboratories International, Inc., Wilmington, MA, USA) were obtained as also described in detail previously (Ramirez et al. 1996). All experimental procedures were approved by the Institutional Animal Care and Use Committee at the Seattle Children’s Research Institute. Mice were deeply anaesthetized with isoflurane (4%) before quick decapitation. Isolated brainstems were then placed in ice-cold aCSF bubbled with carbogen (95% O2–5% CO2). The aCSF contained (in mm): 118 NaCl, 3 KCl, 1.5 CaCl2, 1 MgCl2, 24 NaHCO3, 0.5 NaH2PO4, and 30 d-glucose, pH 7.4. Brainstems were glued rostral end up onto an agar block for mounting into a vibratome (Leica Microsystems, GmbH, Wetzlar, Germany). A single 550–600 μm-thick slice was then taken. Slices were transferred into a recording chamber, continuously superfused with oxygenated aCSF, and maintained at a temperature of 30° ± 0.5°C. To initiate and maintain fictive respiratory rhythmic activity, the potassium concentration of the perfusate was raised from 3 to 8 mm over a period of 30 min. We routinely measured the Inline graphic in the bath before, during and after the exposure to aCSF bubbled with 95% O2 or 95% N2 as described in detail in the study by Hill et al. (2011).

Extracellular population and intracellular current clamp recordings from the preBötC

In the transverse slice preparation, extracellular population recordings were obtained with suction electrodes positioned on the surface of the ventrolateral region containing the preBötC. To obtain a signal containing multi-unit action potential (AP) activity, extracellular signals were amplified 10,000-fold and filtered between 0.25 and 1.5 kHz using an AM instruments (A-M Systems, Sequim, WA, USA) extracellular amplifier (Fig. 1B, top trace). To facilitate the detection of bursts, this signal was rectified and integrated by using an electronic integrator with a time constant of 50 ms (Fig. 1B, middle trace) using home-built equipment. Intracellular current clamp recordings were obtained from respiratory neurons of the contralateral preBötC with the blind-patch technique. The patch electrodes were manufactured from filamented borosilicate glass tubes (Warner Instruments, Hamden, CT, USA 150TF), filled with a solution containing (in mm): 140 potassium gluconate, 1 CaCl2.6H2O, 10 EGTA, 2 MgCl2.6H2O, 4 Na2ATP, and 10 Hepes (pH 7.2). In some cases the intracellular pipettes contained biocytin (4.5 mg ml−1) to allow for the identification of neuron location and morphology. Recordings were low-pass filtered (0–2 kHz, Bessel four-pole filter, –3 dB). Neurons were identified as respiratory neurons by their discharge pattern in phase with the population activity of the contralateral preBötC (Fig. 1B, bottom trace).

Figure 1. In vivo and in vitro measurements of distinct respiratory activities.

Figure 1

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A, a typical example of an EMG recording from a freely breathing anaesthetized mouse. In the top trace is shown the integrated and rectified trace (Int. EMG) of the raw EMG trace (EMG). The inset shows a typical example of a sigh interrupting eupnoea. B, multi-unit recording from a transverse brainstem slice preparation containing the preBötC (preBötC) and the integrated and rectified trace (Int. preBötC) and simultaneous intracellular recording (intra) of an inspiratory preBötC neuron in current clamp. Note that under control conditions the slices can generate two distinct activities (‘fictive eupnoea’ and ‘fictive sighs’).

Voltage clamp recordings of preBötC neurons

Whole cell patch clamp recordings of inspiratory neurons were obtained with a sample frequency of 10 kHz and a low-pass filter setting of 2 kHz. Recordings were made with unpolished patch electrodes, manufactured from borosilicate glass pipettes with a filament (Warner Instruments G150F-4, Warner Instruments, Hamden, CT, USA). The electrodes had a resistance of 3–5 MΩ when filled with the whole cell patch clamp pipette solution containing (in mm): 140 potassium gluconate, 1 CaCl2.6H2O, 10 EGTA, 2 MgCl2.6H2O, 4 Na2ATP, and 10 Hepes (pH 7.2). The patch clamp experiments were performed with a patch clamp amplifier (Multipatch 700B, Molecular Devices, Sunnyvale, CA, USA), a digitizing interface (Digidata 1440A, Molecular Devices), and the software program pCLAMP 10.0 (Molecular Devices). Neurons located at least three to four cell layers (about 80–150 μm) caudal from the rostral surface of the slice were recorded under visual control. Neurons located directly at the slice surface were not examined because their dendritic processes were more likely to be damaged during the preparation than those of neurons located deeper within the slice. Current–response traces were recorded with either off- or online leak subtraction (P/4 protocol), eliminating the linear leak current and residual capacity currents. The 2 mV liquid junction potential was manually subtracted with the amplifier’s pipette offset regulator immediately before establishing the patch clamp configuration. The series resistance was always 80% compensated and regularly corrected throughout the experiments. We emphasize that whole cell voltage clamp recordings from neurons embedded in a functional network are accompanied by difficult space clamp control. This could lead to incorrect values for current amplitudes. Thus, recordings with obvious space clamp were discarded. Consulting equilibrium potential to holding potential relationships, all upward deflections had to be chloride-conducting, inhibitory postsynaptic currents (IPSCs), whereas all downward deflections had to be excitatory postsynaptic currents (EPSCs). EPSCs and IPSCs were analysed by using MiniAnalysis 5.41 (Synaptosft, Inc., Decatur, GA, USA) and statistical analysis was performed with Prism (GraphPad Software, Inc., La Jolla, CA, USA). The synaptic drive current in individual neurons, which occurred in phase with the rhythmic population discharges of the preBötC, was assessed by low-pass filtering the intracellularly recorded current traces (10 Hz) and determining the negative peak amplitude (maximum negative current during the burst). This amplitude was then compared during control conditions in the presence of 100 nm PGE2 and in the presence of 2 μm PGE2 (Fig. 6Aa).

Figure 6. PGE2 has no major effect on synaptic transmission in preBötC neurons.

Figure 6

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A, typical example of an inspiratory preBötC neuron receiving phasic input during eupnoeic and sigh bursts. In rectangle ‘a’ the drive current was determined as the peak negative amplitude of the phasic input after using an offline 10 Hz low pass filter. In rectangle ‘b’ note the incoming EPSCs and IPSCs between two bursts. B, quantification of the drive current, sIPSCs and sEPSCs under control conditions, after exposure to 100 nm and 2 μm PGE2 (n = 5 for each group, *P < 0.05).

Results

PGE2 injections into the preBötC differentially affect sighs and eupnoeic breathing in vivo

In anaesthetized freely breathing CD1 mice (male and female, P9–16, n = 6) we tested the effect of microinjections of three different concentrations of PGE2 (100 nm, 300 nm and 1 μm) into the right preBötzinger complex. The breathing was recorded with EMG recordings from the intercostal muscles (Fig. 1A, for details see Methods). At the lower concentrations we observed an increase in sigh frequency (*P < 0.05, one-way ANOVA, Fig. 2A and B), while the eupnoeic frequency was unaltered (Fig. 2B). In contrast, higher concentrations of PGE2 (1 μm) led to a significant increase in sighs and eupnoea compared to control conditions (*P < 0.05, one-way ANOVA Fig. 2B).

Figure 2. Microinjections of PGE2 into the preBötC increased the frequency of sighs and eupnoeic breathing at different concentrations.

Figure 2

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A, a typical example of the response to microinjections of PGE2 (100 and 300 nm) into the preBötC of a freely breathing mouse. Note the increase of sighs at low concentrations (example trace in A and quantification in B). B, higher concentrations (1 μm) of PGE2 injections increased the frequency of eupnoea and sighs (*P < 0.05).

PGE2 application directly modulates the activity in the preBötC

Next we tested the effects of PGE2 on the preBötC network in the isolated brainstem slice preparation of CD1 mice (male and female, P0–5). In a first set of extracellular experiments, the population activity of the preBötC was recorded with a surface electrode before and after bath application of PGE2. Similar to the in vivo experiments we found a dose-dependent effect of PGE2 on the fictive eupnoeic and sigh activities generated within the slices. At low concentrations (10–100 nm) PGE2 strongly stimulated the frequency of sighs (Fig. 3A and B), without significantly affecting the amplitude, duration or frequency of eupnoeic bursts generated in the preBötC (Fig. 3C). At higher concentrations (2 μm) PGE2 evoked a similar increase in sigh frequency as observed at lower concentrations, but PGE2 had an additional enhancing effect on the eupnoeic frequency compared to control (Fig. 3C, **P < 0.01, paired t test). The effects on both fictive eupnoea and fictive sighs were reversible upon washout.

Figure 3. Bath applications of PGE2 increased the frequency of fictive sighs and eupnoea at different concentrations.

Figure 3

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A, typical extracellular recordings from the preBötC of an isolated brainstem slice preparation in response to bath application of PGE2 (10 nm, 100 nm and 2 μm). B, fictive sigh frequency increased in response to PGE2 at both low (10 nm (n = 8), 100 nm (n = 6)) and high concentrations (2 μm, n = 6). C, only high concentrations (2 μm) PGE2 exposure led to a significant increase in eupnoeic burst frequency compared to control (**P < 0.01, paired t test), while at all concentrations no change in amplitude or duration of the eupnoeic bursts was observed.

PGE2 increases the fictive gasping response of the preBötC to hypoxia

To assess if PGE2 changes the activity of the preBötC during hypoxia, we quantified the response to severely reduced levels of oxygen (95% N2, 5% CO2, Hill et al. 2011) in the absence and presence of different concentrations of PGE2. For these experiments we applied PGE2 for at least 10 min before the measurements. We characterized only one hypoxic exposure per slice. As previously described, exposure to hypoxia leads to a typical biphasic response with an early augmentation and a late depression phase in which the network reconfigures to generate fictive gasping (Fig. 4A, Telgkamp & Ramirez, 1999; Lieske et al. 2000; Peña et al. 2004; Hill et al. 2011). Slices exposed to moderate (100 nm, n = 6) or high (2 μm, n = 6) concentrations of PGE2 (Fig. 4B, C and D) generated gasping activity with significantly increased amplitude and frequency values compared to untreated control slices (Fig. 4D, *P < 0.05, **P < 0.01, one-way ANOVA). Slices exposed to the low concentration (10 nm) did not show a significantly different gasping compared to control slices (n = 8, one-way ANOVA).

Figure 4. PGE2 amplifies gasping activity generated in the preBötC.

Figure 4

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Typical biphasic responses of the preBötC to hypoxia (95% N2, 5% CO2) in a control slice (A) and a slice treated with PGE2 (B, 100 nm). C and D, in the presence of PGE2 (at concentrations ≥100 nm) slices generated an amplified gasping response (with a higher amplitude and frequency) compared to control slices (*P < 0.05, **P < 0.01).

Developmental changes in PGE2 response

To determine if the effect of PGE2 was dependent on the age of the animals we tested a set of slices obtained from animals during their second postnatal week (P7–12, n = 6 for 2 μm, n = 4 for 10–100 nm) and compared them to the data of the slices obtained during the first 5 days of life (n = 6 for 2 μm, n = 14 for 10–100 nm). The increase of the sigh frequency induced by a bath application of a low or high concentration of PGE2 was significantly more pronounced in slices of mice prepared during the first postnatal week (Fig. 5A, two-way ANOVA, Holm-Sidak´s multiple comparison test). By contrast, slices obtained during the second postnatal week revealed only a slight increase at the low concentration (10–100 nm) and a slightly more pronounced effect in response to the high concentration of PGE2 (2 μm) (Fig. 5A). High concentrations of PGE2 significantly increased the frequency of fictive eupnoea in both age groups and no significant difference between the first compared to the second postnatal week was detected (Fig. 5B).

Figure 5. Age dependence of the PGE2 effect on sigh generation.

Figure 5

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A, slices of the first postnatal week showed a strong increase in fictive sigh frequency in response to low (10–100 nm) or high concentrations (2 μm) of PGE2, while slices of the second postnatal week showed a significantly less pronounced increase to low or high concentrations of PGE2. (*P < 0.05, two-way ANOVA, Holm-Sidak’s multiple comparisons test). B, slices of the first and second postnatal week showed a similar significant increase in the frequency after exposure to high concentrations of PGE2 (2 μm).

Spontaneous excitatory and inhibitory postsynaptic currents and the drive current

To test if PGE2 affects synaptic transmission between respiratory neurons we recorded from inspiratory preBötC neurons in voltage clamp to measure the excitatory and inhibitory synaptic events (spontaneous (s)EPSCs and (s)IPSCs). Inspiratory neurons were defined as neurons that received phasic input during the population activity measured with a surface electrode on the preBötC (Fig. 6Aa). We measured sEPSCs and sIPSCs between the rhythmic population bursts before and after adding PGE2 (100 nm and 2 μm) to the bath (Fig. 6Ab and B). As described in detail in the Methods section, we quantified amplitude, decay time and frequency of the EPSCs and IPSCs as an average of 50 events for each condition (control, 100 nm PGE2, 2 μm PGE2, n = 5 for all groups). PGE2 led to a small, but significant reduction of the amplitude of sEPSCs (*P < 0.05, one-way ANOVA) in the presence of 100 nm PGE2, which returned to control values at the high concentration. All other measured parameters did not change when exposed to PGE2 (Fig. 6B, one-way ANOVA). In addition, PGE2 at high or low concentrations did not significantly change the drive current (Fig. 6Aa) compared to control conditions (Fig. 6B).

PGE2 increases intrinsic bursting activity in inspiratory neurons

To test if PGE2 modulates the intrinsic properties of preBötC neurons, we recorded from inspiratory neurons, which discharged in phase with the population burst recorded from the surface of the preBötC in the current clamp configuration (Fig. 7A). In a first set of experiments we tested the response of the neurons within the intact network (n = 9). Most neurons showed a slight depolarization of several millivolts in response to both tested concentrations (100 nm, +4.75 ± 3.10 mV, n = 4; 2 μm, +1.65 ± 1.53 mV, n = 5). However, there was no significant change in the drive potential underlying fictive eupnoea or fictive sighs (Fig. 7A). Next we tested the effect on the neurons in isolation from synaptic inputs. All examined neurons (n = 16) showed either spontaneous tonic firing or intrinsic bursting firing (Fig. 7B, n = 9), when pharmacologically isolated from fast synaptic inputs by the addition of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 20 μm), CPP (10 μm), picrotoxin (5 μm) and strychnine (1 μm) to the bath. Similar to the neurons tested with intact synaptic transmission, PGE2 caused a slight depolarization (2.65 ± 3.00 mV, n = 16). The depolarizations caused by either 100 nm or 2 μm PGE2 were not significantly different in the tonic firing cells or intrinsic bursting neurons.

Figure 7. PGE2 enhances intrinsic bursting properties of inspiratory neurons.

Figure 7

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A, typical example of an inspiratory neuron (intra) firing in phase with the population of the preBötC (int. PreBötC). The drive current was determined after using an offline 10 Hz filter. No significant change in the amplitude of the drive current during the sigh bursts or the eupnoeic bursts was detected in the presence of PGE2. B, intracellular recording of an inspiratory preBötC neuron with intrinsic bursting properties after blocking all fast synaptic transmission (with a cocktail containing: 20 μm CNQX, 10 μm CPP, 5 μm picrotoxin and 1 μm strychnine). Bath application of PGE2 increased the amplitude of the bursts in intrinsic bursting neurons. C, note the increase in bursting frequency in the presence of PGE2. D, quantification of the increase in burst frequency, burst amplitude and AP/burst in intrinsic bursting neurons before and after application of PGE2. *P < 0.05.

In intrinsic bursting cells we found a significant increase in the amplitude and frequency of the bursting after exposure to 2 μm PGE2 (Fig. 7C and D, *P < 0.05, paired t test, n = 9). It needs to be emphasized that bursting was irregular in some cases, and we also observed a substantial variability in the range of bursting in these cells. Thus, the evaluation of the burst frequency over a long period of time may not have been always consistent for all neurons. Previous studies identified two subtypes of pacemaker neurons based on the currents that are critical for the generation of the intrinsic bursts. Neurons that burst depending on the ICAN were termed cadmium sensitive (CS), because bursting was blocked by cadmium, a blocker of calcium currents. Neurons with bursting properties that depend on the persistent sodium current were termed cadmium insensitive (CI) neurons, because their bursting persisted in the presence of cadmium (Thoby-Brisson & Ramirez, 2001; Peña et al. 2004). The stimulating effect of PGE2 was found in CI pacemaker neurons (n = 4). These neurons were sensitive to 10 μm riluzole or insensitive to 200 μm Cd2+. A stimulating effect of PGE2 was also found in CS neurons (n = 3). These neurons were sensitive to 200 μm Cd2+. In two neurons the response to Cd2+ or riluzole was not further tested. Since we only tested the high concentration of PGE2 we were not able to distinguish from these experiments whether a specific cell type was essential for the specific effect of PGE2 on sigh frequency. We therefore conducted additional extracellular experiments to assess the effect of these blockers at the network level.

Differential contribution of ICAN and INap to the PGE2 effect

As mentioned above, it was previously shown that intrinsic bursting properties in the inspiratory neurons of the preBötC can depend either on the persistent sodium current (INap) or on a calcium-activated non-selective cation conductance (ICAN) (Peña et al. 2004). To test if ICAN- or INap-dependent bursting plays a critical role in mediating the effect of PGE2, we performed extracellular experiments and applied PGE2 in the presence of flufenamic acid (FFA, 50 μm), a substance that is known to inhibit the ICAN current (n = 5), or in the presence of riluzole (n = 4), a substance that is known to inhibit the persistent sodium current. For both experimental series, PGE2 was applied first at a concentration that under control conditions specifically stimulated (Fig. 3A) the sigh frequency (100 nm). PGE2 was subsequently applied at a higher concentration (2 μm), which increased additionally fictive eupnoea. In the presence of FFA the slices still showed a significant increase in the sigh frequency in response to application of PGE2 (n = 5, one-way ANOVA, Fig. 8A and C), while in the presence of riluzole (10 μm) sighs were inhibited and could not be stimulated by PGE2 (Fig. 8B and C). At the higher concentration of PGE2 (2 μm), eupnoeic bursting frequency was not significantly increased in the presence of FFA (Fig. 8D), compared to an almost twofold increase in the presence of riluzole (Fig. 8E and F, **P < 0.01, one-way ANOVA). This increase was similar to the effect caused by high concentrations of PGE2 without an additional drug (Fig. 3). In the presence of riluzole and PGE2 (2 μm) the amplitude of eupnoeic bursting decreased compared to control conditions.

Figure 8. Contribution of ICAN and INap to the effects of PGE2.

Figure 8

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A, B and C, low concentrations of PGE2 (100 nm) increased fictive sigh frequency in the presence of FFA (50 μm), while this effect was abolished in the presence of riluzole (10 μm). D, E and F, high concentrations of PGE2 (2 μm) increased eupnoeic bursting frequency in the presence of riluzole, but this effect was not observed in the presence of FFA. *P < 0.05, **P < 0.01.

Discussion

Here we demonstrate that prostaglandin E2 (PGE2) differentially stimulates respiratory activities generated within the preBötzinger complex. Under normoxic conditions, low concentrations (10–100 nm) of this molecule specifically increased the frequency of sighs (augmented breaths), while exposure to higher concentrations of PGE2 (1–2 μm) also increased the frequency of eupnoeic activity in vitro and in vivo (Figs 2 and 3). Prostaglandins affect a variety of other systems, and are activated under both physiological as well as pathophysiological conditions (e.g. Caggiano & Kraig, 1999). In the nervous system these molecules are involved in the production of fever (Scammell et al. 1996; Lazerus et al. 2007), mediation of pain (Ahmadi et al. 2002), modification of behaviour (Tanaka et al. 2012) and synaptic plasticity (Chen & Bazan, 2005; Koch et al. 2010). Indeed, prostaglandins exert diverse effects throughout the body. PGE2 mediates its tissue-specific and cell-specific effects through the activation of EP receptors, which are further classified into the receptor subtypes EP1, EP2, EP3 and EP4 (for a detailed review see Narumiya et al. 1999). These G protein-coupled receptors can increase (EP2 or EP4) or decrease (EP3) intracellular cAMP or increase the internal free calcium concentration (EP1) (Funk et al. 1993; Narumiya et al. 1999). Furthermore, splice variants of the EP3 receptor exist and can activate different signalling pathways. EP3A receptors are coupled to Gi leading to a decrease of cAMP, whereas the EP3B and EP3C receptors are coupled to Gss and act to increase levels of cAMP.

The cell-specific effects can vary between different brain areas. For example PGE2 increases the amplitude of EPSPs by activation of cAMP as a second messenger system in the CA1 pyramidal cells of the hippocampus (Chen & Bazan, 2005), while in cortical pyramidal neurons PGE2 decreases the amplitude of EPSPs via the EP3 receptor (Koch et al. 2010). In the striatum PGE2 enhances GABAergic inputs to dopaminergic neurons of the substantia nigra pars compacta via EP1 signalling (Tanaka et al. 2009), while in the spinal cord PGE2 inhibits glycine-dependent synaptic inhibitory transmission via the EP2 receptor (Ahmadi et al. 2002). PGE2 modulates hyperpolarization-activated currents (Ih, Ingram & Williams, 1996), potassium currents (Chen & Bazan, 2005) and sodium currents (Gold et al. 1996; Natura et al. 2013). Thus, it is conceptually interesting to hypothesize that even within the same area the concerted action of different EP receptors can provide a balance between concurrent excitatory and inhibitory PGE2 effects. The complexity of the effects caused by prostaglandins is an important consideration when interpreting our results in the context of other studies.

To the best of our knowledge this is the first study investigating how PGE2 affects the local circuitry surrounding the preBötC and neurons within the preBötC. The excitatory effects observed in our study are not unexpected, given that the activation of several EP receptors causes an increase in intracellular calcium and cAMP. An increase of cAMP is a well-known, powerful stimulant of breathing (Mironov et al. 1999; Shao et al. 2003; Lieske & Ramirez, 2006b), as was also demonstrated by Ballanyi et al. (1997). The excitatory effects of elevating cAMP (Lieske & Ramirez, 2006b) are similar to the excitatory effect of PGE2 as described in the present study.

Previous studies that also explored the central effects of prostaglandins on breathing obtained results that are partly different from the data presented here (Ballanyi et al. 1997; Tai & Adamson, 2000; Hofstetter et al. 2007; Siljehav et al. 2012). Given the complexity of the modulatory effects caused by prostaglandins such differences may not be surprising, in particular since the other studies employed different approaches and addressed the role of prostaglandins in different contexts. Hofstetter et al. (2007) specifically demonstrated that the respiratory depression caused by hyperoxia in the presence of the cytokine interleukin-1β (IL-1β) was abolished in mice that lacked mPGES1, a gene essential for the synthesis of PGE2. IL-1β also reduced the number of anoxia-induced gasps, a reduction which was also abolished in the gene-targeted mice. Moreover, PGE2 (4 nm) injected into the ventricles induced apnoeas, an effect which was abolished in EP3 knock-out mice (EP3R–/–), but the gasping response in the EP3R–/– mice was not altered. PGE2 also caused an inhibition of respiratory activity in the isolated brainstem–spinal cord preparation, which was abolished in EP3R–/– mice. By contrast we observed that PGE2 application caused an overall excitatory effect on respiration, including the gasping response generated within the preBötC (Fig. 4).

The differences are best explained by the different approaches. Hofstetter et al. (2007) examined the effects of injections into the peritoneum, ventricles and the brainstem–spinal cord, using combinations with other cytokines, while we focused our study on the direct effects of PGE2 on the preBötC. By design, several of the treatments by Hofstetter led to the systemic activation of inflammation cascades. Moreover, the Hofstetter study inferred several of the PGE2 effects, based on different knockout mice in which either the enzyme necessary for the production of PGE2 or specific EP receptors were mutated. Thus, compensatory mechanisms could have been partly induced by the global mutations of enzymes and receptors. There is no question that the Hofstetter study provided interesting and clinically relevant insights into the modulatory and interacting effects of cytokines and prostaglandins. However, the experimental design of their study was more complex and probably involved other areas and additional effects on the respiratory network.

Indeed, this caveat has to be considered also in the context of our study. Although, we are probably the first to examine the effects caused by PGE2 in the transverse slice that contains the preBötC, we cannot exclude the possibility that PGE2 might also have affected mechanisms outside of the preBötC. The transverse slices contain, for example, the nucleus of the solitary tract, Raphe nuclei and various noradrenergic nuclei (Doi & Ramirez, 2008; Ramirez et al. 2012), all of which could influence the prostaglandin responses. Our in vivo injections targeted the preBötC, but the injections typically spread beyond the preBötC (Zanella et al. 2014), and thus probably also affected other areas as well as potentially inputs into the preBötC.

Our study also needs to be considered in the context of the study by Ballanyi et al. (1997) who reported an inhibition of brainstem activity in response to prostaglandin E1 (PGE1) exposure. These authors used the brainstem–spinal cord preparation, which includes areas other than those encompassed by the transverse slice preparation. Moreover, PGE1 has a partly different affinity to the EP receptors compared with that of PGE2. Both molecules bind strongly to EP3 and EP4 receptors at low concentrations but at higher concentrations PGE2 is more selective for the EP1 receptor. In addition PGE1, but not PGE2 binds at low concentrations to IP receptors (the Prostaglandin I2 receptors) (Narumiya et al. 1999). Since the effect on the EP3 and EP4 receptors should be similar for PGE1 and PGE2, the differences might be best explained by the activation of IP receptors by PGE1. PGE1 can also act via the inhibition of the inositol 1,4,5-trisphosphate pathway (Tysnes et al. 1990), which has been shown to be an important regulator of bursting activity in the preBötC (Crowder et al. 2007).

Our discussion clearly illustrates the complexity and diversity of the modulatory effects caused by prostaglandins. These considerations are particularly important when interpreting the action of prostaglandins in a clinical context in which inflammatory processes, cytokines and prostaglandins act on different receptors and second messenger systems that will differently affect different parts of the body, the CNS and even different areas of the respiratory network.

Could different effects reflect physiological vs. pathological states of PGE2 signalling?

Keeping all the caveats in mind, it is tempting to speculate that the differential effects of PGE2 at high and low concentrations could reflect physiological and pathological roles of PGE2 in the regulation of respiration. The effect of low concentrations activating specifically sighs is interesting since this respiratory behaviour has also been associated with the arousal response (Lijowska et al. 1997; Ramirez, 2014). The majority of arousal responses begin with the generation of sighs (Gerard et al. 2002; Wulbrand et al. 2008; Ramirez, 2014). A loss of sighs has also been associated with premature death in mice, associated with lung atelectasis (Koch et al. 2013). In addition, the network that is responsible for the generation of the sighs is also sensitive to hypoxia and facilitates the generation of sighs before it reconfigures to gasping. Thus, an increased activation of sighs associated with slightly elevated COX-2 activity may be associated with an increased alertness that may prepare the organism to adverse conditions (Ramirez, 2014). By contrast, high levels of PGE2 might only be reached during extraordinary circumstances like severe hypoxia or systemic infections that require a full activation of the respiratory network to meet the metabolic demand. Clearly, these are just some possible scenarios that can be considered in the context of an inflammatory response, which will affect many different aspects of the respiratory response as was discussed in the previous section.

Differential modulation of intrinsic membrane properties by PGE2 and the generation of different inspiratory rhythms in the preBötC

The rhythm-generating mechanisms of the preBötC have been under systematic investigation over the past decades and there is general consensus that various synaptic and intrinsic membrane properties contribute to the generation of the respiratory rhythm, even though the relative importance of each of these mechanisms is still a matter of discussion (Ramirez et al. 2012; Feldman, 2013; Richter & Smith, 2014). It is also generally agreed that the neuronal activity generated within the preBötC is controlled by numerous neuromodulators that provide the respiratory network with a high degree of plasticity (Gray et al. 1999; Shao et al. 2005; Doi & Ramirez, 2010). These modulators alter synaptic and intrinsic membrane properties in a complex manner (Shao et al. 2005; Doi & Ramirez, 2010; Ramirez et al. 2011; Viemari et al. 2013). The present study provides additional insights into the neuromodulation of the preBötC by exploring the modulatory effects of PGE2 on the synaptic and intrinsic membrane properties of inspiratory neurons. The specific and differential network effects caused by PGE2 at low and high concentrations indicate that this molecule will not simply lead to a dose-dependent increase in either excitatory synaptic transmission or intrinsic excitability of all respiratory neurons. Supporting this idea we did not observe significant changes in the excitatory or inhibitory synaptic tonic drive (sEPSCs and sIPSCs) after application of low or even high concentrations of PGE2 (Fig. 6C and D). We also know that the majority of respiratory neurons within the preBötC are active during both fictive sighs as well as fictive eupnoeic activity (Lieske et al. 2000; Chapuis et al. 2014); thus we do not favour the hypothesis that the differential effect of PGE2 is caused by the differential activation of different types of neurons. However, this explanation cannot be fully excluded since a very small proportion of neurons within the respiratory network are activated only during sighs and could therefore be the target of low concentrations of prostaglandins (Tryba et al. 2008; Chapuis et al. 2014). However, even though the vast majority of neurons are activated during both respiratory activities, sighs and eupnoeic activities are differentiated by specific differences in their synaptic and intrinsic properties (Lieske & Ramirez, 2006a,b; Koch et al. 2013). In a recent study we demonstrated that genetic ablation of P/Q-type calcium channels (Cav2.1) leads to the selective loss of sighs and a reduction in excitatory synaptic transmission between respiratory neurons (Koch et al. 2013). These differential effects support the possibility that a differential effect on calcium currents could potentially favour the activation of sighs as opposed to eupnoeic activity at low PGE2 concentration. This possibility needs to be further explored by specifically characterizing the PGE2 effects on different calcium currents.

However, the best insights into the differential effects of PGE2 were gained by the use of flufenamic acid and riluzole, substances that are known to affect the calcium-activated non-selective cation current (ICAN) and persistent sodium current (INap), respectively (Peña et al. 2004; Del Negro et al. 2005). Both currents differentially contribute to the bursting generation of respiratory neurons (Thoby-Brisson & Ramirez, 2001; Ramirez et al. 2012), and both types of bursting properties were also affected by PGE2. Low and high concentrations affected gasping, a respiratory activity, which under our experimental conditions depends on the persistent sodium current and not ICAN (Peña et al. 2004). This dependence on INap is explained by the reconfiguration of the respiratory network during gasping. In this state neurons dependent on ICAN are inhibited, while neurons dependent on the INap continue to burst (Peña et al. 2004). The INap has also been associated with the generation of sighs (Viemari et al. 2013), and the low-concentration effect of PGE2 on the sigh was abolished in the presence of riluzole, but not by blocking ICAN with flufenamic acid. Thus, we hypothesize that the low concentrations of PGE2 could exert their effects on sighs and gasping by modulating the persistent sodium current. Sodium channels are known to be modulated by multiple neurotransmitters, including PGE2, acting through cAMP and activation of protein kinase A (PKA) and protein kinase C (PKC) (for detailed review see Cantrell & Catterall, 2001). In neurons of the cortex and hippocampus an activation of the cAMP pathway (via PKA or PKC) results in a decrease of the amplitude of sodium currents (Cantrell et al. 1996; Maurice et al. 2001), while other groups found an increase in the persistent sodium current in neocortical neurons (Astman et al. 1998). Thus, it seems plausible that PGE2 could activate the cAMP pathway at lower concentrations. This activation could amplify the persistent sodium current and modulate the generation of the sigh. However, this hypothesis needs to be investigated in future studies.

By contrast, the eupnoeic effects caused by high concentrations of PGE2 are probably not mediated by the persistent sodium current, since these effects persisted in the presence of riluzole. Instead, these eupnoeic effects could be mediated by PGE2 acting on the ICAN. It is well established that the ICAN plays a critical role in the generation of eupnoeic activity (Peña-et al. 2004; Del Negro et al. 2005; Pace et al. 2007). Moreover, ICAN is very sensitive to changes in the intracellular calcium concentration, which is increased by PGE2 acting on a variety of receptors. Thus, we hypothesize that the effects caused by high concentrations of PGE2 are mediated by the activation of the ICAN current. These effects are possibly mediated by an increased intracellular calcium concentration.

Clearly, our considerations can only be regarded as hypotheses that will require further investigation. It will be particularly interesting to explore how the entirely excitatory effects as described in the present study could become inhibitory when PGE2 is applied into the ventricle or in the context of an inflammatory response as described by other laboratories (Hofstetter et al. 2007). The drastic differences in the modulatory responses of PGE2 as described by different laboratories under different experimental conditions promises to yield fascinating insights into the plasticity of the respiratory network, a network that needs to alter its responsiveness in a very sensitive, and diverse manner to changes in the physiological and pathophysiological conditions. Understanding the details of these modulatory responses will be particularly important for unravelling how inflammation affects the nervous system as it transitions from an adaptive to a maladaptive response. In many disease states, acute inflammatory responses are initially protective, but become detrimental under chronic conditions. The assumption that a neuromodulatory response remains qualitatively the same under different conditions needs to be re-considered as was also illustrated in our recent study in which noradrenaline transitioned from a neuromodulator that regularizes respiratory activity into a modulator that severely destabilizes respiration after exposure to intermittent hypoxia (Zanella et al. 2014). The differential activation of multiple receptor subtypes with their numerous second messenger systems offers a wide range of possibilities to orchestrate such transitions that will dramatically alter network functions in health and disease.

Glossary

aCSF

artificial cerebrospinal fluid

AP

action potential

COX-2

cyclooxygenase-2

EMG

electromyography recordings

FFA

flufenamic acid

ICAN

calcium-activated non-selective cation conductance

INap

persistent sodium current

PGE1

prostaglandin E1

PGE2

prostaglandin E2

preBötC

preBötzinger complex

sEPSCs

spontaneous excitatory post synaptic currents

sIPSCs

spontaneous inhibitory postsynaptic currents

Additional information

Competing interests

None declared.

Author contributions

H.K. and J.-M.R. designed the research, H.K.,C.C., F.P.E., A.D. and A.J.G. performed the research; H.K. and C.C. analysed the data; H.K. and J.-M.R. wrote the paper. All authors approved the final version of the manuscript.

Funding

This work was supported by the National institutes of Health grants R01HL107084 and P01HL090554.

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