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NEUROSCIENCE |
1 Department of Applied Science, The College of William and Mary, Williamsburg, VA 23187-8795, USA
2 Systems Neurobiology Laboratory, Department of Neurobiology, David Geffen School of Medicine at the University of California Los Angeles, Box 951763, Los Angeles, CA 90095-1763, USA
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Abstract |
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(Received 8 November 2006;
accepted after revision 31 January 2007;
first published online 1 February 2007)
Corresponding author C. A. Del Negro: Department of Applied Science, McGlothlin-Street Hall, Room 303, The College of William and Mary, Williamsburg, VA 23187-8795, USA. Email: cadeln{at}wm.edu
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Introduction |
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Shortly after discovering that rhythmogenesis in vitro was independent of reciprocal postsynaptic inhibition (Feldman & Smith, 1989), neurons with voltage-dependent bursting-pacemaker properties were characterized in the preBötC (Smith et al. 1991; Johnson et al. 1994), which were subsequently shown to depend on persistent Na+ current (INaP) (Del Negro et al. 2002a,b). These observations led to the hypotheses that INaP plays a key role in respiratory rhythm generation by: (i) giving rise to an obligatory subpopulation of rhythmically active pacemaker neurons, and; (ii) amplifying excitatory synaptic input in non-pacemaker neurons to promote robust inspiratory bursts throughout the preBötC network (Onimaru et al. 1995; Rekling & Feldman, 1998; Butera et al. 1999b, 2005; Smith et al. 2000; Ramirez et al. 2004; Feldman & Del Negro, 2006). Here, we address these hypotheses and conclude that pacemaker neurons are not obligatory for respiratory rhythm generation and INaP does not amplify synaptic inputs but it does enhance spike frequency.
The biophysical properties of INaP have been studied in synaptically isolated and dissociated preBötC neurons but not in the context of respiratory network function. At postnatal days (P) from P0–15, 5–15% of all preBötC neurons exhibit INaP-mediated bursting-pacemaker activity after synaptic isolation using a cocktail of receptor antagonists (Pena et al. 2004; Del Negro et al. 2005). The INaP bursting mechanism is now well understood (Butera et al. 1999a; Thoby-Brisson & Ramirez, 2001; Del Negro et al. 2002a; Ramirez et al. 2004). Nevertheless, all inspiratory neurons express INaP (Del Negro et al. 2002b; Ptak et al. 2005) and receive rhythmic synaptic excitation when synaptic transmission and network function are intact (Funk et al. 1993). This led to the hypothesis that INaP amplifies synaptic excitation, which putatively augments inspiratory bursts throughout the preBötC during respiratory rhythm generation (Butera et al. 1999b; Smith et al. 2000; Del Negro et al. 2001, 2002a), which we test here.
The obligatory rhythmogenic role of pacemaker neurons and INaP in general is questionable because bath application of the Na+ channel antagonist riluzole (RIL) rapidly blocks INaP and pacemaker activity at low doses (IC50
3 µM) but does not rapidly perturb the frequency of respiratory rhythms in vitro and in situ at doses much greater than 3 µM (Del Negro et al. 2002b, 2005; Paton et al. 2006). However, in addition to blocking INaP, RIL depresses excitatory transmission (Doble, 1996; Wang et al. 2004) and long duration, bath application of RIL causes the XII motor discharge to decline in amplitude (Del Negro et al. 2005).
In full recognition of the pharmacological caveats associated with blocking INaP, we present specific and relevant tests of its role in rhythm generation. First, we tested the role of INaP in synaptic amplification with an intact network. We employed intracellular QX-314 (2 mM) to block Na+ currents in single neurons and measured changes in the inspiratory drive potential. We also bath-applied RIL (10 µM) and low doses of tetrodotoxin (TTX, 20 nM) to measure the contribution of INaP to inspiratory drive potential generation and intraburst spiking.
Second, we tested whether pacemaker neurons were required for rhythm generation by microinjecting RIL and TTX directly into the preBötC. We performed this experiment in the presence and absence of flufenamic acid (FFA, 100 µM); we did this because FFA blocks a Ca2+-activated non-specific cation current (ICAN), which engenders another class of bursting activity in neonates older than P8 (Thoby-Brisson & Ramirez, 2001; Ramirez et al. 2004). This drug-microinjection protocol blocks INaP locally within the preBötC and minimizes effects on INaP in neurons outside of the preBötC, such as XII motoneurons and serotonergic neurons in the raphe that may be critical for rhythm generation in vitro (Pena & Ramirez, 2002; Tryba et al. 2006) and perinatal breathing in infants (Paterson et al. 2006).
Here we show that INaP promotes spiking during inspiratory bursts but does not notably amplify synaptic excitation nor contribute to the underlying inspiratory drive potential. Also, blocking INaP in the preBötC does not stop rhythmogenesis even after attenuating ICAN. We conclude that INaP in preBötC neurons is not critical for respiratory rhythm generation in vitro. However, our results suggest that INaP plays a role in respiratory-related neurons outside the preBötC that ultimately helps to maintain the general level of excitability in the preBötC to ensure rhythmogenesis.
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Methods |
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Neonatal mice were anaesthetized by hypothermia and rapidly decerebrated prior to dissection in normal artificial cerebrospinal fluid (ACSF) containing (mM): 124 NaCl, 3 KCl, 1.5 CaCl2, 1 MgSO4, 25 NaHCO3, 0.5 NaH2PO4, and 30 D-glucose, equilibrated with 95% O2 and 5% CO2 with pH = 7.4. Transverse slices (550 µm thick) containing the preBötC, XII motoneurons and the raphe obscurus (Fig. 1A) were sectioned using a vibrating microslicer. The rostral cut was positioned just rostral to the rostral-most XII nerve roots at the level of the dorsomedial cell column and principal lateral loop of the inferior olivary nucleus, thus the preBötC was at or near the rostral surface (Ruangkittisakul et al. 2006). The caudal cut captured the obex. Slices were placed rostral surface up in a 0.5 ml recording chamber on a fixed-stage microscope equipped with Koehler illumination and perfused with 27°C ACSF at 4 ml min–1. ACSF K+ concentration was raised to 9 mM and respiratory motor output was recorded from XII nerve roots using suction electrodes and a differential amplifier (Fig. 1). In voltage-clamp experiments, CaCl2 was reduced to 0.5 mM and replaced with equimolar MgSO4 in order to block synaptic transmission and Ca2+ currents.
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The standard potassium gluconate patch solution contained (mM): 140 potassium gluconate, 5 NaCl, 1 EGTA, 10 Hepes, 2 Mg-ATP, and 0.3 Na-GTP (pH = 7.3 by KOH). In potassium gluconate experiments, pipette resistance was 3–4 M
and a liquid junction potential of 8 mV was corrected offline. In some cases, 2 mM QX-314, obtained from Sigma (St Louis, MO, USA), was added to the potassium gluconate patch solution in order to block Na+ channels intracellularly. In these experiments, we obtained control measurements using nystatin-perforated patches. Nystatin (250 µg ml–1) was added to the QX-314 patch solution immediately prior to use and was discarded after 120 min.
To analyse miniature synaptic potentials (e.g. Fig. 3), we used a Cs+-based patch solution containing (mM): 110 CsCl, 20 TEA-Cl, 10 NaCl, 1.5 EGTA, 10 Hepes, 0.5 CaCl2.2H2O, 2 Mg-ATP, and 0.3 Na-GTP (pH = 7.3 by CsOH). In experiments using the Cs+-based patch solution, pipette resistance was 3–4 M and a liquid junction potential of 3 mV was corrected offline.
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We recorded miniature EPSPs (mEPSPs) with 1 µM TTX to stop network activity and block INaP, and thus specifically measure the effects of RIL on the presynaptic probability of spontaneous neurotransmitter release. PTX (5 µM) and STR (5 µM) were applied to block inhibitory ionotropic receptors. A Cs+-based patch solution was used to block K+ currents and reduce electrotonic attenuation of synaptic potentials. Neurons were held at –60 mV using bias current. We generated a baseline noise histogram and a Gaussian distribution was fitted to determine the standard deviation (S.D.) of baseline noise. Synaptic events were selected by a threshold-crossing algorithm with detection level to exceed 2 times the S.D., which makes the likelihood of detecting a spurious synaptic event P < 0.05. We tested whether RIL modified the amplitude or period of spontaneous mEPSPs using cumulative probability histograms and the Kolmogorov–Smirnov statistic (K-S test) with minimum probability at P < 0.05.
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Results |
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Testing the role of Na+ currents using intracellular QX-314
To measure the postsynaptic contribution of Na+ currents in drive potential generation, we added 2 mM QX-314 to the potassium gluconate patch solution to block Na+ currents intracellularly in only the recorded neuron while network properties and synaptic transmission remained unaltered. In contrast to higher doses, 2 mM QX-314 has minimal affects on Ca2+ currents (Talbot & Sayer, 1996; Hu et al. 2002), but still blocked action potential generation (Fig. 2C). Drive potentials in control were measured with perforated patch recording (Fig. 2A, left trace) and for one minute after rupturing the patch before QX-314 dialysed the cytosol (Fig. 2A, middle trace). After 8–10 min of whole-cell recording in the absence of Na+ currents (e.g. Fig. 2A, right trace), both the amplitude and area of the drive potential increased to 140 ± 13% and 178 ± 30% of control, respectively (P < 0.01). In contrast, QX-314 had no effect on the onset latency, as determined by the appearance of synaptic potentials emerging from baseline noise prior to the inspiratory XII output (P = 0.6, n = 6, Fig. 2B). These findings suggest that the postsynaptic mechanisms involved in drive potentials remain undiminished, and in fact surprisingly increase following intracellular Na+ channel blockade.
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QX-314 blocks all Na+ currents, so it cannot be used to measure how INaP in particular contributes to drive potentials and regulates intraburst spike activity. We tested these roles of INaP with bath-applied 10 µM RIL, which blocks INaP within 6 min (IC50 = 2.4 µM) (Del Negro et al. 2002b; Ptak et al. 2005). While RIL may attenuate transient sodium currents (Do & Bean, 2003; Ptak et al. 2005), action potential generation is largely unaffected (Fig. 4A (inset) and Del Negro et al. 2002b).
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After 12 min, RIL exposure significantly decreased mEPSP frequency, as shown by a significant rightward shift in the cumulative period histogram (P < 0.05, Fig. 3C), without significant effects on the cumulative amplitude histogram (P > 0.05, Fig. 3B). Thus, 10 µM RIL (12–14 min) attenuated the probability of quantal neurotransmitter release, which is consistent with inhibitory effects on presynaptic neurotransmission, and due to the experimental conditions, this effect cannot be attributed to reduction of INaP.
In rhythmically active slices, 10 µM RIL exposure for 6–8 min significantly reduced the number of spikes per burst to 58 ± 10% of control (from 6.1 ± 1.3 to 3.9 ± 1.3, P < 0.01) but had no effect on inspiratory drive potentials: amplitude and area remained at 103 ± 5% and 96 ± 6% of control (P > 0.4). XII amplitude and area were also unaffected: 102 ± 4% and 103 ± 6% of control, respectively (both P > 0.5). Respiratory frequency remained at 87 ± 9% of control (P = 0.3, n = 4, Fig. 4A).
However, after 15 min (at which time RIL caused presynaptic inhibition, e.g. Fig. 3), 10 µM RIL decreased the amplitude and area of drive potentials to 62 ± 8% and 41 ± 9% of control and further reduced the intraburst spiking to 22 ± 8% of control (all P < 0.05). XII amplitude and area both decreased significantly to 63 ± 7% of control, and respiratory frequency decreased to 60 ± 18% of control (all P < 0.05, n = 4, Fig. 4A).
We used a low concentration of TTX (20 nM) as an alternative agent to evaluate the role of INaP. Figure 4C shows the steady-state current–voltage (I–V) relation after blocking synaptic transmission and Ca2+ channels with a low Ca2+ ACSF containing 200 µM Cd2+. TTX (20 nM) selectively reduced the inwardly rectifying or negative slope region of the I–V curve within 4–6 min (n = 5, Fig. 4C and D). The TTX-sensitive inward current (measured at steady-state during 500 ms-long voltage steps) activated near –60 mV and peaked near –40 mV, indicative of INaP in respiratory neurons (Del Negro et al. 2002a; Rybak et al. 2003; Ptak et al. 2005). Subsequently adding 1 µM TTX to the bath solution caused little additional attenuation (n = 3, Fig. 4C and D) suggesting that INaP is quickly and effectively blocked by 20 nM TTX.
Next, we tested the effects of 20 nM TTX on inspiratory drive potential generation. TTX significantly decreased the number of spikes per burst to 44 ± 8% of control within 4–8 min (from 17.8 ± 3.5 to 8.0 ± 2.5, P < 0.05) but did not affect the amplitude or area of drive potentials, which remained at 100 ± 6% and 94 ± 9% of control, respectively (both P > 0.6). Within 4–8 min, 20 nM TTX had no effect on XII motor output or respiratory frequency (all P > 0.2, n = 5, Fig. 4B). However, after 10–40 min of exposure, 20 nM TTX suppressed spiking activity and ultimately led to rhythm cessation (Del Negro et al. 2005).
Microinjection experiments to test whether INaP and pacemakers are obligatory
To limit the effects of RIL and TTX on respiratory-related neurons situated outside the preBötC, we performed bilateral microinjection experiments that target drug delivery within the preBötC. To verify that our microinjection pipettes were accurately positioned, we first bilaterally microinjected the GABAA receptor agonist muscimol (MUS, 15 µM), which when properly placed stopped the rhythm within 20–60 s. Moving either pipette 90–150 µm from the optimal position failed to block rhythmogenesis, even after 10 min of continuous microinjection (n = 3, Fig. 5).
In every experiment, we first used the control MUS injection protocol (Fig. 5) to verify the correct positioning of the microinjection pipettes. After recovery from MUS, we bilaterally microinjected 10 µM RIL for > 20 min, which had no effect on the amplitude (94 ± 5% of control, P > 0.25), area (90 ± 6% of control, P > 0.09) or period of XII motor output (94 ± 5% of control, P > 0.25). RIL in the preBötC did not destabilize the rhythm; the coefficient of variation (CV) of the period was 0.22 ± 0.03 in control versus 0.21 ± 0.01 during 10 µM RIL microinjection (P = 0.6, n = 5, Fig. 6A).
We employed slices within a developmental period (P0–4) containing exceedingly few, if any, pacemaker neurons that depend on ICAN (Pena et al. 2004; Del Negro et al. 2005). To rule out the unlikely rhythmogenic contribution of ICAN pacemaker neurons, we added 100 µM FFA to the bath solution during bilateral RIL microinjection. FFA was applied for > 20 min; the cumulative RIL microinjection time was > 40 min. Together, FFA and RIL significantly reduced the amplitude and area of the XII motor output to 82 ± 4% and 63 ± 5% of control, respectively (both P < 0.05), but had no significant effect on respiratory period (120 ± 11% of control, P > 0.25) or CV (0.19 ± 0.06, P > 0.25, n = 5, Fig. 6A).
Unlike local application in the preBötC, e.g. Fig. 6A, bath application of 10 µM RIL in conjunction with 100 µM FFA stops rhythmogenesis in vitro (Pena et al. 2004; Del Negro et al. 2005). We replicated this result in the microinjection experiment by subsequently bath-applying 10 µM RIL with 100 µM FFA, in which case the rhythm stopped in 7.4 ± 2.2 min (n
= 5). After 
2 min of rhythm cessation, we applied 0.5 µM substance P (SP), which revived respiratory rhythm in 3 of 5 slices. This suggests that bath application of RIL affects the state of neuronal excitability in the preBötC, but does not cause a fundamental breakdown in the rhythmogenic mechanisms since SP can recover the rhythm in the majority of slices tested. In all cases, XII motor output recovered in washout of > 1.5 h (n
= 5, Fig. 6A).
We repeated the experiment in Fig. 6A using 20 nM TTX instead of RIL. To preclude any possible contribution due to ICAN pacemaker neurons, we bath-applied 100 µM FFA for > 15 min before bilaterally microinjecting 20 nM TTX directly into the preBötC for > 10 min. TTX microinjection in the presence of FFA had no significant effect on the amplitude and area of XII motor output (P > 0.4, n = 4). Respiratory period increased to 133 ± 9% compared to the control following recovery from MUS (P < 0.05, n = 4), but did not stop after > 10 min of TTX bilateral microinjection in the presence of FFA. After washout from local 20 nM TTX, we microinjected 1 µM TTX directly into the preBötC, which caused rhythm cessation within 1 min and verified the ability of TTX to quickly penetrate the tissue (n = 2, Fig. 6B).
Microinjection experiments outside the preBötC to test the rhythm-generating role of INaP in neurons outside the preBötC
In the presence of bath-applied FFA, bath-applied RIL caused rhythm cessation whereas local RIL application within the preBötC did not. To explain these observations, we hypothesized that INaP in areas outside the preBötC is important for maintaining rhythmogenesis. One possible target is serotonergic neurons in the raphe, which help maintain preBötC neuron excitability. We tested whether blocking INaP in the raphe region would decrease excitability in the preBötC to perturb or prevent rhythmogenesis. We locally microinjected 10 µM RIL along the midline in the raphe obscurus in the presence of 100 µM FFA; XII motor output stopped in 3–6 min (n = 6, Fig. 7). In all cases, XII motor output recovered fully in washout.
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Discussion |
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We first analysed whether transient and persistent Na+ currents contribute to synaptic amplification by blocking their actions using intracellular application of QX-314 and found that inspiratory drive potentials actually increased. The inability of 20 nM TTX and RIL to attenuate drive potentials within 8 min of drug application supports the notion that INaP does not contribute to drive potential generation. Both drugs blocked INaP within 6 min of drug application (Fig. 4C and Del Negro et al. 2005). While we applied both TTX and RIL for > 19 min, after 12 min these agents perturbed other cellular and network properties that are important in rhythmogenesis (see above). That QX-314 caused a statistically significant increase in the drive potential may reflect the removal of a Na+ channel-mediated shunt, but this remains to be tested.
In contrast, we found that blocking INaP with either 10 µM RIL or 20 nM TTX decreased the number of spikes per burst by 40–50%, suggesting that INaP promotes intraburst spike output. This is consistent with the expression of INaP predominantly at the soma and axon initial segment where it assists in spike initiation and promotes repetitive firing (Lee & Heckman, 2001; Astman et al. 2006; Palmer & Stuart, 2006) and in proximal dendrites (Mittmann et al. 1997). Additionally, INaP is measurable in acutely dissociated respiratory neurons that are extensively denuded of dendritic processes (Ptak et al. 2005), which confirms INaP expression in the soma/axon initial segment of preBötC neurons.
To explore the rhythmogenic role of INaP in preBötC neurons, we used microinjection protocols to deliver drugs directly into the preBötC. Putatively rhythmogenic preBötC neurons are interneurons (Gray et al. 1999) that form synapses onto somata and dendrites within the preBötC (Guyenet & Wang, 2001; Wang et al. 2001; Stornetta et al. 2003). The extent to which INaP is expressed in preBötC neuron dendrites is presently unknown. However, drugs microinjected directly into the preBötC will affect INaP at both somatic and dendritic sites contained within the preBötC.
The fact that RIL and TTX did not stop the rhythm when microinjected in the preBötC in the presence of 100 µM FFA indicates that INaP and associated pacemaker properties are nonessential for rhythmogenesis. Since RIL was applied directly within the preBötC, its low solubility (Doble, 1996) was not a factor that would prevent RIL from penetrating the rhythm-generating network. This fact is underscored by identical microinjection protocols in the raphe that did stop the rhythm within 6 min, confirming that RIL did affect the neurons at the injection site.
In contrast to RIL, TTX reduced the frequency of XII motor output in the presence of 100 µM FFA. This reduction in frequency is most likely attributable to the attenuation of spike-generating currents caused by 20 nM TTX after several minutes of exposure (Fig. 4B, 19 min; and Del Negro et al. 2005). Despite these additional effects, 20 nM TTX ultimately failed to block rhythmogenesis. Changing the concentration of microinjected TTX from 20 nM to 1 µM quickly caused rhythm cessation and confirmed that TTX rapidly affected preBötC neurons.
The fact that RIL and TTX reduced intraburst spiking by 50% without affecting drive potentials appears paradoxical. In a highly interconnected network, such as in the preBötC (Rekling et al. 2000), reductions in neuronal firing would be expected to diminish the presynaptic neurotransmitter release underlying postsynaptic drive potential generation, and thus reduce the magnitude of the drive potential. However, intrinsic burst-generating currents that are activated by synaptic input could compensate for significant reductions in presynaptic transmitter release, as long as postsynaptic activity is sufficient to activate these currents. The INaP-independent ionic mechanisms for synaptic amplification, and their activation mechanisms, remain to be determined.
In its generalized form, the pacemaker hypothesis posits that pacemaker neurons are obligatory for rhythmogenesis (Smith et al. 1991). More recently, the hypothesis that either INaP- or ICAN-mediated pacemakers can drive the rhythm independently has been proposed (Ramirez et al. 2004). Accordingly, to stop rhythm generation both pacemaker phenotypes must be blocked.
The hybrid pacemaker-network version of the pacemaker hypothesis (Smith et al. 1991, 2000) is founded on an essential core population of INaP pacemaker neurons but the role of INaP extends to non-pacemaker neurons where it is postulated to amplify excitatory synaptic drive and enhance inspiratory burst generation. Accordingly, the dynamic interactions of non-pacemaker and pacemaker neurons control respiratory frequency (Butera et al. 1999a, 2000; Best et al. 2005).
Our results are not consistent with either the generalized or hybrid versions of the pacemaker hypothesis. First, INaP did not amplify synaptic excitation per se but did enhance production of action potentials. Second, the rhythm continued in the presence of ICAN and INaP antagonists (Fig. 6), which stop all known pacemaker-neuron activity (Del Negro et al. 2002b, 2005; Pena et al. 2004). The observation that bath application of both INaP and ICAN antagonists can stop respiratory rhythmogenesis in vitro has been used to support the generalized pacemaker hypothesis (Pena et al. 2004; Paton et al. 2006; Tryba et al. 2006). However, this interpretation is flawed for the following reasons. (i) The rhythm can be revived with either SP or AMPA (see Fig. 6A and Del Negro et al. 2005; Feldman & Del Negro, 2006). This indicates that the fundamental mechanism of rhythmogenesis was intact after pharmacological elimination of pacemaker properties; all that was required was a sufficient boost in excitability via exogenous agents. (ii) Bath application of drugs affects all neurons within the slice preparation, including key populations external to the preBötC such as raphe neurons, respiratory premotoneurons and XII motoneurons. The effects of these antagonists on other populations of respiratory-related neurons can readily explain their effects on rhythm without the need to invoke any changes in preBötC neuron function.
For example, blocking INaP in raphe obscurus neurons in the presence of 100 µM FFA caused rhythm cessation. The raphe neurons project to and appear to increase neuronal excitability in the preBötC (Di Pasquale et al. 1994; Al-Zubaidy et al. 1996; Bou-Flores et al. 2000; Pena & Ramirez, 2002). This experiment can potentially explain why, in the presence of FFA, blocking INaP with RIL locally applied to the preBötC had no effect on respiratory frequency, whereas the subsequent addition of RIL to the bath stopped the rhythm within 7 min (a timeframe that indicates RIL was still primarily acting on INaP). We propose that INaP in raphe, and possibly other respiratory-related neurons exclusive of the preBötC can influence excitability within the rhythmogenic network.
Our study illustrates that data obtained following bath-application of drugs to slices must be cautiously interpreted and need multiple controls. In particular, the network level effects of bath-applied drugs cannot be casually attributed to a small subpopulation of preBötC pacemaker neurons embedded in active slices that contain numerous respiratory interneurons as well as premotor and motoneurons.
Pacemaker neurons with as yet undiscovered biophysical mechanisms, i.e. neither INaP nor ICAN, for bursting may play a role generating respiratory rhythm, but the existence of such neurons remains to be demonstrated. Otherwise, the accumulating evidence suggests that pacemaker neurons are not obligatory for respiratory rhythm generation (Del Negro et al. 2002b, 2005; Kosmidis et al. 2004). Instead, we suggest the group-pacemaker hypothesis (Rekling & Feldman, 1998; Feldman & Del Negro, 2006) as a viable alternative explanation. In this framework, periodic inspiratory bursts result from recurrent excitatory connections within the preBötC. Intrinsic currents will naturally play a role in recurrent excitation. We postulate that an inward current that can be directly evoked synaptically via intracellular signalling would be advantageous in this regard compared to a voltage-dependent current like INaP, because its burst-generating function need not depend on the baseline membrane potential. In contrast, INaP only influences respiratory neurons in a voltage window between –60 and –40 mV: INaP remains in a deactivated state when the membrane potential is too low and its contribution steadily diminishes at depolarized potentials due to steady-state inactivation (Butera et al. 1999a; Del Negro et al. 2001, 2002a; Ptak et al. 2005). The activation properties of an inward current directly coupled to synaptic input need not be subject to voltage fluctuations and would therefore be well equipped to amplify synaptic excitation and promote robust and reliable inspiratory bursts.
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x dt) of the inspiratory drive potential.
