Sodium and Calcium Current-Mediated Pacemaker Neurons and Respiratory Rhythm Generation

I_NaP-mediated pacemaker neurons

Figure 1 depicts typical I_NaP-mediated bursting in a mouse pre-BötC inspiratory neuron (Smith et al., 1991; Johnson et al., 1994; Koshiya and Smith, 1999; Thoby-Brisson and Ramirez, 2001; Del Negro et al., 2002a,b). Here, we used patch solution containing 0.1 mm EGTA. Respiratory drive potentials occurred at membrane potentials below the I_NaP activation threshold (Fig. 1A, left) (Rybak et al., 2003). Depolarization evoked I_NaP and caused ectopic bursts in the intervals between XII discharges (Fig. 1A, middle). Intrinsic bursting was confirmed by its persistence after blocking synaptic transmission with CNQX, APV, BIC, and STR in either 9 mm (Fig. 1A, right) or 3 mm (Fig. 1B) K⁺ concentration in the ACSF. Bursting was voltage dependent. Depolarizing the baseline membrane potential using bias current (I_a) caused the cell to move from quiescence to bursting, and further depolarization increased burst frequency (Fig. 1B). However, 20 μm RIL blocked I_NaP-mediated bursting within 90 s, and no amount of depolarizing I_a could restore it (Fig. 1C). All rhythmically active preBötC neurons with stable baseline membrane potentials and overshooting spikes were tested for pacemaker activity and are included in our database. I_NaP-mediated bursting was detected in eight of 178 P0-P5 preBötC neurons (4.5%) and in three of 54 P8-P10 preBötC neurons (5.6%), which was not significantly different (Fisher exact test, p = 0.3).

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Figure 1.

I_NaP-mediated bursting in a mouse preBötC neuron. A, Respiratory activity at -60 mV baseline membrane potential (V_M). ACSF contained 9 mm [K⁺]. Ectopic bursts emerged after adding 50 pA of bias current (I_a). Intrinsic bursting after CNQX, APV, BIC, and STR eliminated respiratory network activity (XII). B, Intrinsic bursting in the same cell as A after reducing ACSF [K⁺] to 3 mm. Baseline V_M is -55 mV. C, Adding 20 μm RIL blocked bursting, which could not be restored after 60-90 s by depolarization via I_a. Calibration applies to all traces.

Calcium current-dependent pacemaker neurons

Bursting in preBötC neurons in mice >P5 that depends on I_Ca is a recent discovery (Thoby-Brisson and Ramirez, 2001). In the past, we never observed this phenotype in P0-P5 rats or mice (Del Negro et al., 2002a), so here we recorded in juvenile mice ages P8-P10 to see whether developmental effects could explain this disparity. After identifying a pacemaker neuron in CNQX, APV, PTX, and STR (Fig. 2A), we applied Cd²⁺ to test whether I_Ca was required. We found four I_Ca-dependent pacemaker neurons in 54 neurons sampled (7.5%) during P8-P10, whereas we found only one I_Ca-dependent pacemaker neuron in 178 neurons sampled during P0-P5 (0.6%), which indicates significantly higher prevalence of bursting cells in the older neonates (Fisher exact test, p = 0.01). Of the 54 preBötC neurons recorded at P8-P10, 40 were recorded with 10 mm EGTA patch solution (Thoby-Brisson and Ramirez, 2001) and 14 with 0.1 mm EGTA patch solution. I_Ca-dependent bursting neurons in these older neonates made up 7.5% of the sample regardless of EGTA concentration (n = 3 of 40 and 1 of 14, respectively), which suggests that slow calcium buffering by 10 mm EGTA does not influence pacemaker activity (Fisher exact test, p = 0.43). The I_Ca-dependent pacemaker neurons often showed ramp-like trajectories during the interburst interval, i.e., expiration, and a strong inspiratory discharge that was enhanced by depolarization (Fig. 2A). Bursting continued in CNQX and RIL but ceased in Cd²⁺ (Fig. 2A,D), which demonstrates dependence on I_Ca. Bursting was also blocked by 10 μm FFA (Fig. 2B), which suggests that a Ca²⁺-activated nonspecific cationic current (I_CAN) (Gogelein et al., 1990; Guinamard et al., 2004) carries significant burstgenerating inward current in addition to I_Ca.

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Figure 2.

I_Ca-dependent bursting in mouse preBötC neurons (P8). A, Respiratory discharge from a baseline V_M of -60 mV; adding 30 pA depolarized the cell and strengthened ramp-like V_M trajectory and respiratory drive potentials and spike discharge. Intrinsic bursting continued in CNQX, APV, PTX, and STR. Cd²⁺ blocked bursting but not action potentials (inset). B, A similar experiment, but here the bursting was blocked by 10 μm FFA. C, Bursting behavior in CNQX, APV, PTX, and STR was subjected to depolarizing bias current (I_a). D, RIL at 20 μm did not block bursting but Cd²⁺ did (same cell as C). I_a pulses were used to evoke action potentials in RIL plus Cd²⁺ conditions to ensure that the cell could still generate spikes. E, Burst period from the cell in C (circles) and the voltage-dependent I_NaP pacemaker neuron from Figure 1 B (squares), plotted versus I_a.

I_Ca-dependent bursting was extremely rare in P0-P5 mice (1 of 178; 0.6%), in agreement with Pena et al. (2004). In a P2 mouse we blocked bursting in this one neuron using 200 μm FFA and then added 20 μm RIL, which additionally hyperpolarized the cell by 2-3 mV and thus suggests that this neuron expressed I_NaP in addition to I_CAN.

Unlike I_NaP-mediated bursting, I_Ca-dependent bursting was not voltage dependent. For example, the pacemaker neuron in Figure 2C was subjected to increasing levels of I_a but maintained a ∼4 s burst period (Fig. 2E, filled circles). Figure 2E further compares this cell with I_NaP-mediated voltage-dependent bursting from Figure 1B (open squares). Nevertheless, I_a increased the number of action potentials per burst. For example, depolarization increased the mean number of spikes per burst from 21 to 37 in Figure 2A (in the context of network activity) and from 4 to 7 in Figure 2C (in the isolated pacemaker neuron after CNQX, APV, PTX, and STR).

RIL applications in vitro

We applied RIL for 30 min and monitored respiratory motor output in P0-P5 slices from rats and mice (Figs. 3, 4, 5). RIL at 10 μm reduced XII amplitude (Fig. 3, black triangles) but did not change the mean period (Figs. 3, 5, gray circles) (for n = 3 experiments, Wilcoxon test, p = 0.6). RIL at 10 μm also caused cycle-to-cycle variability in the period that was noticeable but not statistically significant (K-S test, p = 0.08).

RIL at 20 μm decreased XII amplitude monotonically and caused periodic fluctuation but did not change the mean period until after the XII amplitude decreased to baseline, which occurred at 40 min. Afterward, the period rapidly increased (Fig. 3, note the upward thrust of gray circles in the row labeled 20 μm) and within a dozen cycles rhythmic output ceased altogether. Similar results were obtained at RIL concentrations from 50 to 200 μm: motor output disappeared after a precipitous increase in period, which always coincided with XII amplitude reaching barely detectable levels (Fig. 3). Before the abrupt cessation of rhythm, the mean period for n = 3 experiments at each RIL concentration was not significantly different from control (Wilcoxon tests, all p > 0.05). RIL consistently caused periodic fluctuations, which were statistically significant at 50 and 200 μm (K-S test, p < 0.01).

Higher RIL doses accelerated the rate of decline in motor output. The mean time required to abolish XII output decreased sigmoidally with an EC₅₀ value of 32 μm, whereas I_NaP in rats and mice was blocked by much lower doses (EC₅₀ = 3 μm) and within 9 min (Fig. 4) (Del Negro et al., 2002a). This suggests that either respiratory rhythm did not require I_NaP and RIL abolished motor output via some other effect or that I_NaP was required but that RIL affected rhythmogenic neurons only at high doses (or in prolonged exposures).

The second scenario might arise if RIL has difficulty penetrating the slice preparation. To evaluate this, we measured the effects of 20 μm RIL on I_NaP recorded in preBötC neurons from rats and mice at various depths. We previously reported rapid blockade (<9 min) of I_NaP by RIL in neurons within 125 μm of the surface (Del Negro et al., 2002a). Here, we recorded neurons 250-360 μm deep and applied descending voltage ramps that inactivate the fast Na⁺ current responsible for action potentials (but do not completely inactivate I_NaP) every 20 s during RIL application (Del Negro et al., 2002a). Using a cesium-based patch solution in neurons that satisfied our criteria for adequate voltage clamp, a region of negative slope was always present in the steady-state I-V curve because of I_NaP (Del Negro et al., 2002b). RIL always blocked this negative slope region in <9 min (n = 7) such that the I-V curve was monotonic with positive slope between -60 and -40 mV. Under these conditions, voltage-dependent I_NaP bursting is impossible (Smith et al., 1975). In three cells, the time course of I_NaP blockade could be fitted by an exponential decay function with a time constant of 2 min (Fig. 5). Figure 5 also plots the mean respiratory period computed from three slice experiments like Figure 3, which shows that the mean respiratory period was undiminished within the time frame needed for 20 μm RIL to block I_NaP at depths ≥250 μm.

Low doses of TTX

Low doses of TTX preferentially antagonize I_NaP in respiratory neurons (Koizumi and Smith, 2003). We found that 20 nm TTX reversibly abolished respiratory rhythm in 20-40 min in 15 mouse slices (P0-P5) tested (Fig. 6A). An average of the last three to five respiratory bursts showed that XII amplitude decreased 62%, and frequency declined 94% before the rhythm ceased (Fig. 6C). TTX at 20 nm hyperpolarized respiratory neurons by 5-10 mV, which is consistent with blockade of a Na⁺ current active at rest, such as I_NaP.To determine whether 20 nm TTX also affected spike generation, we applied 20 ms step currents from a baseline membrane potential of -60 mV to evoke action potentials (during the expiratory phase). Rheobase increased from 277 ± 177 pA in control to 399 ± 187 pA in TTX (≥30 min exposure to 20 nm TTX) (p < 0.001; n = 9) (Fig. 6B,C). Spike amplitude declined in TTX. Changes in rheobase required more passive charging of the membrane potential to reach spike threshold, and this passive charging progressively encompassed a greater percentage of the total spike amplitude in TTX. Therefore, we defined the active fraction of the evoked spike as the difference between the maximum spike amplitude and the peak of the passive membrane response. Active fraction decreased from 67 ± 10 mV in control to 29 ± 13 mV in 20 nm TTX (p < 0.0005; n = 9) (Fig. 6B,C). All of the changes induced by 20 nm TTX were statistically significant by Wilcoxon signed rank or paired t tests and were reversible in washout (Fig. 6C). In one preparation, 20 nm TTX completely abolished spikes (data not shown).

Because 20 nm TTX hyperpolarized preBötC neurons, increased rheobase and decreased spike amplitude, we posited that TTX-induced decreases in excitability might be responsible for respiratory rhythm cessation, rather than (highly) selective attenuation of I_NaP. After at least 2 min of quiescence, we attempted to recover network function in the presence of 20 nm TTX by adding the excitatory neuropeptide SP (0.5-2 μm). SP revived the rhythm in five of nine preparations tested (Fig. 7). The recovered rhythm in SP and TTX conditions was stable for 5-10 min and then gradually became unstable and ceased after 40-60 min exposure to TTX. Respiratory rhythm recovered fully in washout (Fig. 6A).

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Figure 7.

SP at 2 μm restores respiratory rhythm in a mouse slice exposed to 20 nm TTX. TTX hyperpolarized this inspiratory neuron by 7 mV; thus, 220 pA of depolarizing bias current (I_a) was applied to restore baseline V_M to -60 mV (20 nm TTX, 5 min). Additional depolarizing I_a was required to maintain V_M of -60 mV by the time TTX abolished rhythmic activity (20 nm TTX, 15 min). After the rhythm ceased, adding 2 μm SP to the bath revived it. The amplitude of the cellular drive potentials and XII amplitude recovered in SP, but spike discharge did not occur with baseline V_M at -60 mV (attributable to increases in rheobase, see Results and Fig. 6).

FFA and RIL coapplication experiments

Although I_Ca-dependent pacemaker neurons are extremely sparse in P0-P5 mice, one could argue that during bouts of RIL (e.g., Figure 3), a small number of RIL-insensitive pacemaker neurons sustains the rhythm (Pena et al., 2004). We tested this possibility in FFA and RIL coapplication experiments. In P0-P5 mouse slices, first we applied 100 μm FFA, which did not affect XII frequency or amplitude (Fig. 8A,B) but at the cellular level reversibly reduced (but did not abolish) respiratory drive potentials and spike amplitude (Fig. 8B). After washout, we coapplied 100 μm FFA and 20 μm RIL, which blocked respiratory rhythm within 5 min; the last cycle of cellular and XII activity is shown in Figure 8B. After more than 2 min of quiescence in the presence of FFA and RIL, adding 0.5-2 μm SP to the bath restored rhythmic activity: XII output resembled the first FFA washout, but respiratory drive potentials were significantly attenuated (n = 5 slices). We were also able to rescue the rhythm after it ceased in 100 μm FFA and 20 μm RIL using low concentrations of bathapplied AMPA (500 nm; n = 2).

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Figure 8.

Rhythm generation in the presence of 100 μm FFA and 20 μm RIL. A, Continuous segments of the experiment showing control, FFA, recovery, and then FFA and RIL coapplication, as well as FFA plus RIL plus SP conditions in a mouse slice. Rhythm cessation in FFA plus RIL was rescued by SP. B, Examples of cellular respiratory drive and XII output from A are plotted with greater time resolution. Broken line facilitates XII amplitude comparisons.

I_NaP pacemaker neurons

RIL blocks bursting and I_NaP in preBötC neurons within minutes at low doses (EC₅₀ = 3 μm), whereas it abolishes respiratory rhythm in slices only after long exposures or when concentration is >10 μm (Del Negro et al., 2002a). RIL caused periodic fluctuation at all concentrations, thus I_NaP and pacemaker neurons probably help stabilize network rhythmicity. The time to block XII output was dose dependent (EC₅₀ = 32 μm, corresponding to 35 min for XII discharge to disappear), which is an order of magnitude higher than the EC₅₀ value of 3 μm to block I_NaP within 9 min in neurons recorded at depths up to 350 μm in slices. The disparity in dose and time dependence suggests that the effects of RIL that abolish respiratory rhythm include more than just blocking I_NaP.

I_NaP-mediated bursting in preBötC neurons does not appear to be developmentally regulated because these cells were found at ∼5% in P0-P5 and P8-P10 cohorts. Is 5% a reasonable estimate of the fractional content of I_NaP bursting neurons in the preBötC? Pena et al. (2004) reported 16-29% I_NaP pacemaker neurons in P1-P15 mice. However, these authors preferentially selected neurons that exhibited pacemaker-like characteristics in the cell-attached mode (Pena et al., 2004). Their sampling favors neurons that discharge many spikes per XII cycle and more frequently exhibit pacemaker properties after application of CNQX and other blockers, which significantly overestimates the prevalence of I_NaP pacemaker neurons. Moreover, because they reported that the likelihood of finding pacemaker neurons of any type in a given slice was <10%, then the 16-29% figure cannot be representative of their actual relative prevalence. In contrast, we sampled preBötC neurons without regard to activity pattern in cell-attached mode. All rhythmic neurons were tested for pacemaker properties in the presence of CNQX, APV, BIC/PTX, and STR. We avoided using low Ca²⁺ solution to block synaptic transmission because it also blocks I_CAN and Ca²⁺-dependent K⁺ current and can induce silent cells to burst (Pena et al., 2004). Therefore, we estimate that the actual fraction of I_NaP pacemaker neurons in the preBötC is closer to 5%.

Calcium-dependent pacemaker neurons

We detected only one I_Ca-dependent pacemaker neuron of 178 neurons (0.6%) during P0-P5, whereas we found them 7.5% of the time at P8-P10, which is significantly different. This suggests that either I_Ca (Onimaru et al., 1996, 2003; Elsen and Ramirez, 1998) or I_CAN are developmentally regulated and that Cd²⁺-sensitive bursting behavior emerges predominantly after P5. Independent reports from our group and Pena et al. (2004) show that these cells increase in numbers by an order of magnitude after P6. Despite differences in sampling protocols, these two reports estimate the fraction of I_CAN pacemaker neurons to be between 7.5 and ∼9% in juvenile mice.

I_Ca is necessary for bursting in these neurons because Cd²⁺ blocks pacemaker activity (Thoby-Brisson and Ramirez, 2001). However, the voltage-independent nature of the bursting suggests that Ca²⁺ activates I_CAN, which dominates the burst-generating mechanism. TRPM4- or TRPM5-like ion channels engender I_CAN and are blocked by 30-100 μm FFA (Teulon, 2000; Launay et al., 2002; Montell et al., 2002; Guinamard et al., 2004; Moran et al., 2004). We abolished bursting with 10-200 μm FFA, which is consistent with a role for TRPM4/5-mediated I_CAN (Schiller, 2004). FFA also blocks gap junctions (Srinivas and Spray, 2003; Ye et al., 2003), enhances K⁺ currents (Stumpff et al., 2001), with EC₅₀ < 50 μm so that doses >100 μm could have side effects that impact respiratory network activity (Pena et al., 2004).

Regardless of age, I_Ca-dependent pacemaker neurons showed very strong inspiratory bursts in control and FFA always attenuated drive potentials (Figs. 2A, 8 B). These data suggest that I_CAN contributes significantly to inspiratory bursts even at young ages when the actual fraction of cells with I_CAN pacemaker properties is extremely low.

Low doses of TTX depress excitability in preBötC neurons

Low doses of TTX simultaneously diminished the frequency and amplitude of XII motor output, whereas RIL primarily decreased XII amplitude. TTX (20 nm) hyperpolarized preBötC neurons, increased spike threshold and rheobase, and significantly diminished (or abolished) action potentials. These effects decrease neuronal excitability and could ultimately cause the cessation of respiratory rhythm independent of any direct effects on bursting properties. Unlike TTX, RIL does not significantly alter spike threshold or rheobase at concentrations that block I_NaP (Del Negro et al., 2002a).

Despite the general depression of excitability caused by 20 nm TTX, SP added to the bath restarted respiratory rhythm in most slices. SP depolarizes synaptically isolated rhythmogenic preBötC neurons by closing K⁺ channels and activating a TTX-insensitive linear Na⁺ current, but SP does not induce a region of negative slope conductance nor induce intrinsic bursting behavior in pre-BötC neurons (Gray et al., 1999; Pena and Ramirez, 2004). Here, SP was applied in the presence of TTX. Therefore, spiking properties and I_NaP would remain attenuated, and we conclude that depolarization of preBötC neurons by SP was directly responsible for restarting rhythmic activity, and not the restoration of pacemaker properties.

Rhythm generation in the presence of FFA and RIL

Similar to 20 nm TTX, coapplication of RIL and FFA suppressed respiratory rhythm. Again, the rhythm could be rescued with SP or low concentrations of AMPA, which depolarize cells and boost excitability but do not directly cause pacemaker properties (Gray et al., 1999; Pena and Ramirez, 2004). SP presumably compensates for the hyperpolarization caused by the loss of I_NaP and I_CAN, which suggests that the ability of I_NaP and I_CAN to give rise to bursting-pacemaker activity in a subset of isolated neurons is not essential for rhythmogenesis. Rather, we conclude that normally I_NaP and I_CAN contribute to rhythmogenesis by enhancing the general excitability of the network and promoting inspiratory burst generation in all respiratory preBötC neurons without requiring bursting pacemaker activity.

Emergent network properties

Our data suggest that pacemaker neurons per se are not essential to generate the respiratory rhythm, but that I_NaP and I_CAN contribute to rhythmogenesis by promoting excitability. We favor an emergent network mechanism of respiratory rhythmogenesis such as the group pacemaker (Rekling et al., 1996a,b, 2000; Rekling and Feldman, 1998) in which pacemaker neurons can be embedded but are not obligatory for rhythm generation. We posit that recurrent synaptic excitation in preBötC neurons triggers inward currents carried by I_NaP and I_CAN, thereby amplifying depolarization through a positive feedback cycle (a little synaptic excitation leads to a lot of neuronal output). I_NaP and I_CAN are present in cells without pacemaker activity and serve to boost membrane potential from baseline to suprathreshold levels in response to synaptic activity. Recurrent excitation causes cascades of synaptic activity in cultured spinal cord networks, which are enhanced by intrinsic cellular currents and excitability (Streit, 1993; Streit et al., 2001; Darbon et al., 2002). Respiratory neurons use burst-promoting currents like I_NaP and I_CAN, but the majority of cells are not pacemakers because they do not burst unless synaptically activated. In the absence of I_NaP and I_CAN, we conclude that a compensatory boost in excitability depolarizes constituent neurons closer to spike threshold so that excitatory synaptic drive can still evoke the inspiratory burst without the amplification normally provided by I_NaP and I_CAN.

We propose that inspiratory burst termination results from a collection of intrinsic cellular properties such as I_NaP inactivation (Del Negro et al., 2002b; Rybak et al., 2003), I_CAN deactivation, recruitment of calcium-dependent potassium channels, or electrogenic ion pumps (Ballerini et al., 1997; Darbon et al., 2003). These cellular processes can activate based on Na⁺ and Ca²⁺ accumulation during inspiration. Burst termination does not require coupling, whereas network burst initiation is impossible without the excitatory coupling (Rekling et al., 1996b; Rekling and Feldman, 1998).

Finally, neurons rostral to the preBötC may contribute to rhythm generation (Mellen et al., 2003; Onimaru and Homma, 2003) and some have pacemaker properties (Ballanyi et al., 1999). Because these neurons are not contained in slices, we cannot predict how such cells might contribute to the final pattern of respiratory activity when the preBötC is embedded in the more extensive brainstem respiratory network.