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Abstract

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

The role of AMPA receptors (AMPARs) in generation and propagation of respiratory rhythm is well documented both in vivo and in vitro, whereas the functional significance of NMDA receptors (NMDARs) in preBötzinger complex (preBötC) neurons has not been explored. Here we examined the interactions between AMPARs and NMDARs during spontaneous respiratory rhythm generation in slices from neonatal rats in vitro. We tested the hypothesis that activation of NMDARs can drive respiratory rhythm in the absence of other excitatory drives. Blockade of NMDARs with dizocilpine hydrogen maleate (MK-801, 20 μm) had a negligible effect on respiratory rhythm and pattern under standard conditions in vitro, whereas blockade of AMPARs with NBQX (0.5 μm) completely abolished respiratory activity. Removal of extracellular Mg2+ to relieve the voltage-dependent block of NMDARs maintained respiratory rhythm without a significant effect on period, even in the presence of high NBQX concentrations (≤ 100 μm). Removal of Mg2+ increased inspiratory-modulated inward current peak (II) and charge (QI) in preBötC neurons voltage-clamped at −60 mV by 245% and 309%, respectively, with respect to basal values. We conclude that the normal AMPAR-mediated postsynaptic current underlying respiratory drive can be replaced by NMDAR-mediated postsynaptic current when the voltage-dependent Mg2+ block is removed. Under this condition, respiratory-related frequency is unaffected by changes in II, suggesting that the two can be independently regulated.

Glutamate is the major fast excitatory neurotransmitter underlying respiratory rhythm generation. AMPAR and NMDAR antagonist microinjections in vivo in adult mammals suggest a synergistic role of these receptors in the transmission of inspiratory drive to motoneurons. Microinjection of either the AMPAR antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxalline (NBQX) or the NMDAR antagonist d(–)-2-amino-7-phosphonoheptanoic acid (AP-7) into the phrenic motor nucleus decreases the amplitude of phrenic nerve bursts in rats. However, simultaneous blockade of both receptors decreases the amplitude in a synergistic way (Chitravanshi & Sapru, 1996). Although NMDARs and AMPARs coexist in respiratory rhythm generation-related areas, only AMPAR-mediated transmission is essential for rhythm generation and propagation both in vivo in adult rat (Connelly et al. 1992; Chitravanshi & Sapru, 1996) and cat (Anderson & Speck, 1999), and in vitro in neonatal rat (Greer et al. 1991; Funk et al. 1993). Moreover, in vitro preparations from neonatal mutant mice lacking the NMDAR1 subunit generate a rhythm that is indistinguishable from that obtained from neonatal wild-type mice, demonstrating that NMDARs are not essential for respiratory rhythm generation or drive transmission in the neonate (Funk et al. 1997). However, this does not mean that NMDARs are superfluous. In in vitro preparations generating respiratory rhythm, while the NMDAR antagonist dizocilpine hydrogen maleate (MK-801) does not perturb spontaneous respiratory burst frequency, bath application of NMDA produces a dose-dependent increase in respiratory frequency (Greer et al. 1991; Funk et al. 1993). Furthermore, in vivo, anaesthetized cats breathe normally after systemic administration of MK-801, but subsequent vagotomy produces apneusis (Foutz et al. 1988, 1989; Feldman et al. 1992).

Complicating our understanding of the contribution of NMDARs to rhythm generation is its voltage dependence: at resting membrane potentials (≤−60 mV), currents through activated NMDARs are substantially attenuated by Mg2+ in physiological concentrations (0.8–1.2 mm), but as the membrane depolarizes, the Mg2+ blockade is relieved. Here we examined the effects of removing NMDAR blockade during perturbations of AMPAR-mediated transmission on rhythmic activity of preBötC neurons and integrated hypoglossal nerve (∫XIIn) activity in a neonatal rat medullary slice preparation. We found that under conditions where the voltage-dependent Mg2+ block of NMDARs is relieved, substantial currents can pass through NMDARs sufficient to drive the rhythm when AMPARs are blocked. Moreover, even though in the absence of Mg2+ there is a 4-fold increase on preBötC neuron inspiratory-modulated inward current peak (II), respiratory frequency remained unaffected. We show that the NMDAR can substitute, after removing its voltage-dependent block due to Mg2+, for the AMPAR glutamatergic transmission normally underlying respiratory pattern generation in the in vitro slice preparation.

Methods

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

Slice preparation

Experiments were performed on neonatal rat transverse brainstem slices that generate respiratory-related motor output (Smith et al. 1991). The Office for the Protection of Research Subjects, University of California Research Committee approved all protocols. Briefly, neonatal rats (0–3 days old) were anaesthetized with isoflurane, decerebrated and the neuroaxis was isolated. The cerebellum was removed and the brainstem sectioned serially in the transverse plane using a VT-1000 Vibratome (Vibratome, St Louis, MO, USA) until neuroanatomical landmarks, i.e. nucleus ambiguus and inferior olive, were visible. A transverse slice (550 μm) containing the preBötC was cut. The dissection was performed in an artificial cerebrospinal fluid (ACSF) containing (mm): 128 NaCl, 3 KCl, 1.5 CaCl2, 1 MgSO4, 23.5 NaHCO3, 0.5 NaH2PO4 and 30 glucose, bubbled with 95% O2–5% CO2 at 27°C. The slice was transferred to a 1 ml recording chamber and anchored using a platinum frame and a grid of nylon fibres. The chamber was mounted to a fixed-stage microscope and perfused with ACSF (6 ml min−1).

Rhythmic respiratory-related motor output was recorded from the XIIn using fire-polished glass suction electrodes and a differential amplifier. To obtain a robust and stable rhythm, ACSF K+ concentration was elevated to 9 mm and slices were perfused for 30 min before any experimental manipulation. XIIn activity was amplified, bandpass filtered (0.3–1 kHz), rectified and integrated (τ= 20 ms; ∫XIIn). For 0 mm Mg2+ ACSF, 1 mm MgSO4 was removed. Only one experiment was performed per neuron per slice.

Drugs, obtained from Sigma Chemical Co. (St Louis, MO, USA), were bath applied at the following concentrations: 1 μm tetrodotoxin (TTX), 5 μm bicuculline, 2 μm strychnine, 0.001–100 μm NBQX in a cumulative way, 20 μm MK-801. We used 20 μm MK-801 to specifically block NMDA receptors and avoid non-specific effects (Rothman, 1988; Wooltorton & Mathie, 1995). dl-Threo-β-benzyloxyaspartate (TBOA, 100 μm) was obtained from Tocris (Ellisville, MO, USA).

Patch-clamp recording

Inspiratory neurons from preBötC were visualized using infrared-enhanced differential interference contrast videomicroscopy. Whole-cell patch-clamp recordings were performed using an Axopatch 200A amplifier (Axon Instruments) for voltage-clamp and current-clamp experiments. Electrodes were pulled from borosilicate glass (o.d., 1.5 mm; i.d., 0.86 mm) on a horizontal puller. Electrodes were filled with solution containing (mm): 140 potassium gluconate, 5 NaCl, 10 Hepes, 0.1 EGTA, 2 Mg-ATP, and 0.3 Na3-GTP (pH 7.3). Input resistance (Rin) was determined from the current–voltage relationship generated by slow voltage-ramp commands (∼10 mV s−1) in the linear region negative to −50 mV. Cell capacitance (CM) was determined from the integral of the transient capacitive current (IC, leak subtracted) evoked by 15 ms hyperpolarizing voltage steps (ΔVM), using the formula CM=∫ICVM. Series resistance (Rs) was then calculated from the decay-time constant (τ) of IC since τ∼RSCM in voltage-clamp, where τ is the estimated exponential IC decay time. An acceptable voltage-clamp requires Rin≥ 10 ×RS. Neurons failing to meet this criterion were discarded. RS averaged 19.2 ± 0.9 MΩ (n= 25) and was compensated to 11.0 ± 0.6 MΩ via analog feedback. RS compensation was applied without whole-cell capacitance compensation in order to continuously monitor τ and ensure stationary voltage-clamp conditions.

Electrophysiological signals were acquired digitally at 4–20 kHz using pCLAMP software and a Digidata 1320 AD/DA board (Axon Instruments) after low-pass filtering. Igor Pro (Wave Metrics, Inc., OR, USA), Chart (http://www.ADInstruments.com) and Microsoft Excel were used for data analyses.

Inspiratory-modulated inward current peak (II) was collected from cycles obtained during 3 min recording of steady state activity and averaged. Steady state for each experimental condition was considered after 10 min. Values were normalized against control. Synaptic charge (QI) was computed from the integral of the envelope of II measured at −60 mV.

Excitatory postsynaptic currents were elicited by 100 μm glutamate (IGlu) applied locally with a pressure ejection system (Picospritzer II, General Valve Corp, Fairfield, NJ, USA). Inspiratory neurons were synaptically isolated by 1 μm TTX, and 5 μm bicuculline and 2 μm strychnine to abolish the contribution of spontaneous currents mediated by activation of GABAA and glycine receptors. Pressure ejection pipettes were standard unpolished patch-electrodes positioned at a distance of 30–40 μm from the recorded neuron. The pressure applied ranged between 10 and 20 p.s.i. and the time for each application was 5 ms.

Results are expressed as means ±s.e.m. ANOVA and Student's t test were used when appropriate.

Results

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

AMPARs are obligatory for respiratory rhythm generation; NMDARs are not necessary

Bath application of NBQX at increasing concentrations: (i) decreased ∫XIIn peak amplitude in a dose-dependent manner (EC50= 0.07 μm, n= 4, Fig. 1A); II peak recorded in voltage-clamp at −60 mV (EC50= 0.02 μm, n= 4, Fig. 5A), and; (ii) increased the period (EC50= 0.13 μm, Fig. 1A). Concentrations of NBQX between 0.1 and 0.5 μm eliminated inspiratory currents and hence XIIn activity (Fig. 1B).

image

Figure 1. AMPARs are obligatory for respiratory rhythm generation; NMDARs are not necessary A, antagonism of AMPARs with NBQX produced a dose-dependent decrease of the ∫XIIn amplitude (EC50= 0.07 μm) and an increase in period (EC50= 0.13 μm). Antagonism of NMDAR with MK-801, did not significantly affect ∫XIIn amplitude or period. B, representative II from a preBötC neuron and ∫XIIn discharge recorded from a neonate rat rhythmic slice in standard conditions. Bath application of 0.1–0.5 μm NBQX completely abolished respiratory activity at the system and single unit levels. C, bath application of 20 μm MK-801 alone did not significantly affect respiratory activity at the system and single unit levels. (*P < 0.05; **P < 0.01.)

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image

Figure 5. In 0 mm Mg2+ MK-801 shifted the NBQX dose–response curves for II and period to values similar to those in the presence of Mg2+ A, in 0 mm Mg2+ (^) bath application of NBQX at increasing concentrations decreased the II peak recorded from neurons voltage-clamped at −60 mV (EC50= 0.001 μm, n= 4, note that in the absence of NBQX, 0 mm Mg2+ increased II 3.4 ± 0.8 times with respect to control). II from preBötC neurons was measurable at NBQX concentrations in which II was no longer measurable in control conditions (•). In the absence of Mg2+ and the presence of MK-801 (□), lower concentrations of NBQX were necessary to decrease II peak at −60 mV (EC50= 0.1 μm, n= 4). NBQX dose–response curve for II peak in the presence of MK-801 is similar to the curve in control conditions (EC50= 0.02 μm, n= 4). B, in 0 mm Mg2+ bath application of NBQX at increasing concentrations increased period (EC50= 64.65 μm). The EC50 for period was 500 times greater with respect to the EC50 in control conditions (EC50= 0.13 μm). Bath application of MK-801 increased period to values close to control conditions (EC50= 0.12 μm). C, analysis of synaptic charge (QI) reveals the contribution of different components. Values were normalized against control (QI_ctrl= 1). See Results for description. (*P < 0.05; **P < 0.01.)

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Bath application of MK-801 (20 μm) did not significantly affect ∫XIIn peak amplitude (Fig. 1A, n= 13), the II peak and synaptic charge (QI) in voltage-clamp at −60 mV (n= 9, Figs 1C and 2A) or the mean period (from 7.4 ± 1.2 s in control conditions to 8.5 ± 1.1 s, n= 13, Fig. 1A).

image

Figure 2. NMDARs are present but not passing current in preBötC neurons in standard in vitro conditions A, black trace: II from a preBötC neuron voltage-clamped at −60 mV. Gray trace: II from same neuron in the presence of 20 μm MK-801. MK-801 did not significantly affect II amplitude. B, black trace: II from a preBötC neuron voltage-clamped at +40 mV. Note at this voltage II is outward. Dark grey trace: II from same neuron in the presence of 20 μm MK-801. Relief of voltage-dependent Mg2+ blockade unmasked a large component of NMDAR contributing to II. C, black trace: IGlu elicited by local pressure ejection of 100 μm glutamate in a synaptically isolated preBötC neuron voltage clamped at −60 mV. Gray trace: IGlu elicited with same parameters but in the presence of 100 μm TBOA. Glutamate uptake blockade significantly increased II peak and area. D, dark grey trace: IGlu in 100 μm TBOA. Light grey trace: IGlu in 100 μm TBOA and 20 μm MK-801. In the presence of TBOA, a large fraction of IGlu is NMDAR mediated. In this and subsequent figures, data were normalized against control (control = 1, dashed line), except in D in which data were normalized against values in TBOA. (*P < 0.05; **P < 0.01; ***P < 0.001.)

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NMDARs contribute to II when Mg2+ blockade is removed by depolarization

We questioned whether AMPARs dominate respiratory drive under control conditions due to suppression of NMDAR currents resulting from the latter's voltage-dependent Mg2+ block. In order to eliminate this block, we voltage-clamped preBötC neurons at +40 mV (due to a reversal potential of ∼0 mV, at this voltage II is outward). Subsequent bath application of MK-801 decreased the II peak by 25% (P < 0.05, n= 5, Fig. 2B) and decreased QI by 56% with respect to basal conditions (P < 0.001, Fig. 2B). Thus when the voltage-dependent Mg2+ block is removed, a substantial NMDAR-component contributes to II.

Inhibition of glutamate uptake unmasks a substantial population of NMDARs in preBötC neurons

The current elicited by local pressure ejection of 100 μm glutamate in synaptically isolated (with bath-applied TTX) preBötC neurons (IGlu) was measured in voltage clamp at −60 mV. The non-transportable glutamate uptake inhibitor TBOA (100 μm) was then bath-applied. TBOA increased IGlu by 110% (P < 0.05) and increased QGlu by 520% (P < 0.001) (n= 4, Fig. 2C). To measure the contribution of NMDAR-mediated current to this increase we added MK-801 to the bath. MK-801 reduced both IGlu and QGlu by 40% (P < 0.05) and 45% (P < 0.05), respectively, with respect to IGlu in the presence of TBOA alone (n= 4, Fig. 2D).

Endogenous NMDAR activation affects ∫XIIn amplitude and area, II and QI, but not period

In order to investigate the effects of endogenous NMDAR activation at the network level, i.e. XIIn motor output, we removed the NMDAR Mg2+ blockade. Thus, after recording basal rhythmic activity, control ACSF (containing 1 mm Mg2+) was replaced by an ACSF lacking Mg2+, i.e. 0 mm Mg2+. Under control conditions, the period of ∫XIIn was 6.3 ± 1.2 s (n= 4); 0 mm Mg2+ ACSF did not significantly affect the period (5.5 ± 0.7 s, n= 4) but significantly increased ∫XIIn amplitude and area 28% (P < 0.05) and 43% (P < 0.05), respectively (Fig. 3A and B, n= 4). Subsequent bath application of MK-801 (during 0 mm Mg2+ ACSF perfusion) abolished the effects induced by 0 mm Mg2+ ACSF and brought ∫XIIn amplitude, area and period values back to control levels (Fig. 3B).

image

Figure 3. Endogenous NMDAR activation affects ∫XIIn amplitude and area, II and QI, but not period A and B, perfusion of slices with 0 mm Mg2+ ACSF increased ∫XIIn amplitude and area 28% and 43%, respectively, with respect to basal rhythmic activity (Control). ∫XIIn period in control conditions was 6.3 ± 1.2 s (n= 4) and in 0 mm Mg2+ ACSF was 5.5 ± 0.7 s. Bath application of MK-801 brought ∫XIIn amplitude, area and period values back to control. ∫XIIn period in this condition was 5.8 ± 0.3 s. C, in preBötC neurons voltage-clamped at −60 mV and perfused with 0 mm Mg2+ ACSF, II peak and QI increased 245% and 309%, respectively, with respect to II in control conditions. Representative traces: black: control; grey: 0 mm Mg2+. (*P < 0.05; **P < 0.01.)

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Compared to control ACSF, 0 mm Mg2+ ACSF significantly increased II peak and QI in preBötC neurons voltage-clamped at −60 mV by 245% (P < 0.01, n= 3, Fig. 3C) and 309% (P < 0.01), respectively.

NMDARs drive the respiratory rhythm in the absence of AMPARs

In 0 mm Mg2+ ACSF, bath application of NBQX at increasing concentrations decreased ∫XIIn amplitude (EC50= 0.1 μm, n= 4), decreased II peak recorded from neurons voltage-clamped at −60 mV (EC50= 0.001 μm, n= 4, Fig. 4A and 5A) and did not significantly affect period until very high concentrations (≥ 100 μm, EC50= 64.7 μm, Fig. 5B). There was a 500-fold increase in the NBQX EC50 for period with respect to the EC50 in control ACSF. Thus, in 0 mm Mg2+ ACSF, ∫XIIn discharges and II were measurable at NBQX concentrations that abolished II in control ACSF. Concentrations as high as 100 μm of NBQX were unable to block either II or rhythmic XIIn activity (n= 4, Figs 4A and 5A). To test whether after blockade of AMPARs the remaining II was attributable to active NMDARs, we simultaneously bath-applied MK-801 (20 μm) along with increasing concentrations of NBQX (0.01–0.5 μm). In the presence of MK-801, lower concentrations of NBQX produced the same decrease in ∫XIIn amplitude (EC50= 0.18 μm, n= 4, Fig. 4B) and in II peak at −60 mV (EC50= 0.1 μm, n= 4, Figs 4B and 5A), as well as the same increase in period (EC50= 0.12 μm, Fig. 5B). In the presence of MK-801, 0.5 μm NBQX abolished II, and thus XIIn activity (Figs 4B and 5A).

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Figure 4. NMDARs drive the respiratory rhythm in the absence of AMPARs In 0 mm Mg2+ ACSF, ∫XIIn discharges and II from preBötC neurons were measurable at NBQX concentrations in which II was no longer measurable in basal conditions. A, bath application of NBQX at increasing concentrations decreased the ∫XIIn amplitude but did not affect period. Concentrations as high as 100 μm of NBQX were unable to block either the II in preBötC neurons or the ∫XIIn activity (n= 4). II peak remained virtually unchanged in 1–100 μm NBQX. B, in the presence of MK-801, lower concentrations of NBQX were necessary to decrease ∫XIIn amplitude and II peak, as well as to increase period. NBQX at 0.5–1 μm abolished II.

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Analysis of QI in 0 mm Mg2+ unmasks several components

Under control conditions AMPARs are the main carriers of QI with a small contribution of NMDARs. To quantify the contribution of NMDARs, we computed QI in the steady state of several experimental conditions and normalized those values against QI under control conditions (QI,ctrl= 1, Fig. 5C). Bath application of MK-801 reduced QI to 0.89 ± 0.07, meaning that QI,NMDAR= 0.11. In 0 mm Mg2+, QI increased to 4.09 ± 0.5. We assumed that removal of the voltage-dependent Mg2+ blockade activated NMDARs that were silent under control conditions. Further bath application of MK-801 eliminated the NMDAR-mediated fraction (QI,total,NMDAR), reducing QI to 2.78 ± 0.6, still substantially larger than QI,ctrl. We assumed that this anomalous increase in QI is attributable to non-specific effects of Mg2+ removal, such as an increase of neurotransmitter release from presynaptic terminals, increasing AMPAR- and other neurotransmitter receptor-mediated currents. In 0 mm Mg2+ and 50 μm NBQX, QI was 2.07 ± 0.34. We assume that, in this case NBQX blocked QI,AMPAR and a fraction of QI,non-specific mediated by AMPAR, then the QI carriers were QI,total,NMDAR and QI,non-specific, non-AMPAR.

In summary, in standard conditions AMPARs are the main carriers of QI, with a small contribution of NMDARs. A large fraction of NMDARs do not pass current due to the Mg2+ block. In the absence of Mg2+ that large fraction of NMDARs is unmasked and, in the presence of NBQX, became the major QI carrier.

NMDARs drive the respiratory rhythm in 3 mm K+

Our slices exhibit a robust rhythm in 9 mm K+ and are silent in 3 mm K+ ACSF under standard conditions (data not shown). We questioned whether the additional II generated by NMDARs in the absence of Mg2+ would be enough to drive the respiratory rhythm in 3 mm K+. After recording activity in control ACSF (with 9 mm K+ and 1.5 mm Mg2+), we washed in a test ACSF with 3 mm K+ and 0 mm Mg2+. This increased II peak and QI in preBötC neurons voltage-clamped at −60 mV by 100% (P < 0.05, n= 6, Fig. 6C) and by 170% (P < 0.05), respectively, and increased ∫XIIn amplitude and area by 30% (P < 0.01, n= 6) and 34% (P < 0.05), respectively. Under control conditions, the period was 5.5 ± 1.26 s (n= 6); test ACSF increased the period to 13.6 ± 1.06 s (P < 0.05, n= 6).

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Figure 6. NMDARs drive the respiratory rhythm in 3 mm K+ A, II and ∫XIIn discharges from a preBötC neuron voltage-clamped at −60 mV in standard conditions. Lowering the extracellular [K+] to 3 mm in 0 mm Mg2+ ACSF increased the peak II but slowed the rhythm. Increasing the [Mg2+] to basal values abolished the rhythm. B, same as in A. In 3 mm K+, 0 mm Mg2+ ACSF, bath application of MK-801 abolished the rhythm. C, summary of the effects of lowering extracellular [K+] to 3 mm in the absence of Mg2+. (*P < 0.05; **P < 0.01.)

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Bath replacement with 3 mm K+, 1.5 mm Mg2+ ACSF (n= 3, Fig. 6A) or bath application of MK-801 (n= 3, Fig. 6B) completely abolished II and rhythmic XIIn activity.

Discussion

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

Under control conditions in respiratory rhythmic slices from neonatal rats, NMDARs in preBötC neurons appear activated during inspiration but do not pass significant amounts of current due to their voltage-dependent block. However, NMDARs can significantly contribute to or even solely drive the respiratory rhythm in these slices in situations where the voltage-dependent block is substantially attenuated.

NMDARs are not required for prenatal development of respiratory networks (Funk et al. 1997). In medullary slice or en bloc brainstem–spinal cord preparations from neonatal rodents under baseline conditions, NMDARs are not required for generation of respiratory rhythm or motor output, yet exogenous application of NMDA produces a robust response (Greer et al. 1991; Funk et al. 1993, 1997). Furthermore, removal of extracellular Mg2+ enhances inspiratory currents in preBötC neurons, suggesting that endogenous NMDAR activation can enhance the discharge normally due to currents through AMPARs (Pierrefiche et al. 1991).

We suggest that during each inspiratory cycle only a small fraction of preBötC neuron NMDARs are sufficiently depolarized to remove the Mg2+ block. How big a depolarization is needed to remove the Mg2+ block? That depends on NMDAR subunit composition. NR1/2A (composed of NR1 and NR2A subunits) and NR1/2B receptors are more strongly inhibited at hyperpolarized potentials by Mg2+ than NR1/2C or NR1/2D receptors (Kuner & Schoepfer, 1996). In recombinant systems, inclusion of NR3A with NR1 and NR2 in heteromultimeric channels reduces the sensitivity to Mg2+ block and results in a smaller unitary conductance than NR1/NR2 channels. Consistent with that, in NR3A-deficient mice, NMDA-evoked currents of cortical neurons are larger than in wild-type littermates (Das et al. 1998; Sasaki et al. 2002).

Single-cell RT-PCR analysis reveals that NR2A, NR2B and NR2D are expressed in similar amounts in preBötC neurons, XII motoneurons and neurons from the nucleus of the solitary tract (NTS) in young rats, whereas NR3A is expressed in all preBötC neurons but only in one-third of XII motoneurons and NTS neurons (Paarmann et al. 2005). We suggest that under control conditions, the depolarization achieved during the initial phase of each inspiratory burst relieves the Mg2+ block of NR3A-containing receptors, which are less sensitive to the voltage-dependent blockade. Since these receptors have a small unitary conductance, their contribution to QI is not significant. Depolarizing a neuron or removing the extracellular Mg2+ unmasks the fraction of NMDARs that under control conditions are presumably active, i.e. bound by glutamate, but not passing current. This fraction must contain NR1/NR2A and NR1/NR2B receptors that are strongly blocked near resting membrane potentials by Mg2+ at physiological concentrations.

An interesting observation is that inhibition of glutamate uptake enhances both the AMPAR- and the NMDAR-mediated components of IGlu in preBötC neurons voltage-clamped at −60 mV. We suggest that increased glutamate accumulation in the synapse increases the AMPAR-mediated depolarization of preBötC neurons sufficiently to remove the voltage-dependent block of NMDARs (MacDonald & Nowak, 1990) allowing them to pass current.

In the absence of Mg2+, NBQX concentrations higher than 1 μm decreased ∫XIIn amplitude while II peak and period remain virtually unchanged. We suggest that the AMPAR/NMDAR ratio is higher in XIIn motoneurons than in preBötC neurons, making them more sensitive to AMPA blockade.

Though in standard control conditions NMDARs are not the major charge carrier for II, the Ca2+ influx they provide could contribute to activation of much larger inward currents such as the Ca2+-activated mixed cationic current (ICAN) that is present in all preBötC neurons and are hypothesized to be an important intrinsic burst-generating current (Rekling & Feldman, 1998; Pena et al. 2004; Del Negro et al. 2005).

Implications for respiratory rhythm generation

The group-pacemaker hypothesis posits that preBötC inspiratory neurons mutually interconnected by glutamatergic synapses initiate inspiration by generating a population burst of activity arising from a recurrent network with positive feedback (Rekling & Feldman, 1998). Here we show that, in the absence of AMPAR-mediated synaptic transmission, NMDARs can provide the excitatory drive necessary to initiate and propagate the inspiratory burst. Under our experimental conditions, NMDARs in preBötC neurons can replace AMPARs in mediating excitatory interactions since: (i) Both receptors are coexpressed in preBötC neurons (at present we do not know if they have similar somatodendritic or synaptic distribution; Paarmann et al. 2000); (ii) NMDAR current slope in the absence of Mg2+ is similar to that of AMPARs; and (iii) NMDAR activation can recruit a set of intrinsic conductances resembling those activated by AMPARs, and also induce pacemaker-like membrane potential oscillations (Grillner & Wallen, 1985). These currents are not identical, however. A striking difference is that NMDARs are permeable to Ca2+ whereas in preBötC neurons, AMPARs almost exclusively contain R-edited GluR2 (Paarmann et al. 2000) and are mainly permeable to Na+ (authors' unpublished observations) but not Ca2+. This would suggest that Ca2+ entry through synaptic receptors is not playing a critical role in rhythmogenesis, which is of considerable interest because the Ca2+ buffering of these neurons appears to be limited (Alheid et al. 2002). We suggest that the major source of Ca2+ entry in preBötC neurons is through voltage-gated Ca2+ channels activated during action potentials (C. Morgado-Valle and J. L. Feldman, unpublished data).

Removal of extracellular Mg2+ increased the II and the ∫XIIn amplitude but slightly decreased the period. In contrast, lowering the ACSF [K+] to 3 mm in the absence of Mg2+ increased the II and ∫XIIn amplitude but almost tripled the period. Thus, an increase in II does not necessarily result in an increase in frequency. This would suggest that II affects the amplitude of the motor output whereas frequency depends on the level of excitability of preBötC neurons.

Clinical relevance

Our findings may have clinical relevance. Patients with hyperventilation syndrome (HVS), a breathing pattern disorder characterized by bouts of inappropriately high ventilation associate with elevated frequency, often have significant hypomagnesaemia (Fehlinger & Seidel, 1985; Durlach et al. 1997). Patients with Rett's syndrome also have frequent episodes of hyperventilation; supplemental dietary Mg2+ ameliorates these episodes (Egger et al. 1992). Our work suggests a potential causal link since decreased levels of Mg2+ could increase respiratory output (inappropriately) that is driven by the preBötC by mechanisms described above.

In summary, NMDARs are not necessary for respiratory rhythmogenesis under standard in vitro conditions. However, they can, after removing their voltage-dependent block due to Mg2+, substitute for AMPAR-mediated glutamatergic transmission normally underlying respiratory pattern generation (at least in our experimental conditions). Moreover since II can be modulated independent of frequency and recurrent excitation is necessary for rhythm generation, network connectivity is an essential element underlying respiratory rhythmogenesis. Since Mg2+ levels can affect neuronal plasticity (Slutsky et al. 2004), an additional role of Mg2+ in breathing could be to modulate respiratory plasticity (Feldman et al. 2003), essential for adaptation of breathing to changing demands.

References

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix
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Appendix

  1. Top of page
  2. Abstract
  3. Methods
  4. Results
  5. Discussion
  6. References
  7. Appendix

Acknowledgements

This research was supported by NIH Grant HL-40959. C.M.-V. is a Parker B. Francis Fellow in Pulmonary Research (Francis Families Foundation, Kansas City, MO, USA).