Sodium and calcium current-mediated pacemaker neurons and respiratory rhythm generation

Abstract

The breathing motor pattern in mammals originates in brainstem networks. Whether pacemaker neurons play an obligatory role remains a key unanswered question. We performed whole-cell recordings in the preBotzinger Complex in slice preparations from neonatal rodents and tested for pacemaker activity. We observed persistent Na+ current (I(NaP))-mediated bursting in approximately 5% of inspiratory neurons in postnatal day 0 (P0)-P5 and in P8-P10 slices. I(NaP)-mediated bursting was voltage dependent and blocked by 20 mum riluzole (RIL). We found Ca2+ current (I(Ca))-dependent bursting in 7.5% of inspiratory neurons in P8-P10 slices, but in P0-P5 slices these cells were exceedingly rare (0.6%). This bursting was voltage independent and blocked by 100 microm Cd2+ or flufenamic acid (FFA) (10-200 microm), which suggests that a Ca2+-activated inward cationic current (I(CAN)) underlies burst generation. These data substantiate our observation that P0-P5 slices exposed to RIL contain few (if any) pacemaker neurons, yet maintain respiratory rhythm. We also show that 20 nm TTX or coapplication of 20 microm RIL + FFA (100-200 microm) stops the respiratory rhythm, but that adding 2 mum substance P restarts it. We conclude that I(NaP) and I(CAN) enhance neuronal excitability and promote rhythmogenesis, even if their magnitude is insufficient to support bursting-pacemaker activity in individual neurons. When I(NaP) and I(CAN) are removed pharmacologically, the rhythm can be maintained by boosting neural excitability, which is inconsistent with a pacemaker-essential mechanism of respiratory rhythmogenesis by the preBotzinger complex.

Figure 3.

Effects of RIL on respiratory rhythm in rat slice preparations. Increasing concentrations of RIL plotted from top to bottom. Respiratory period (gray circles) and XII amplitude (black triangles) are plotted versus time. RIL was applied for 30 min in each bout. Period is scaled from 0-24 s (left ordinate), and XII amplitude is scaled uniformly (right ordinate) for each experiment in arbitrary normalized units.

Figure 5.

Effects of RIL on I_NaP and XII respiratory period. I_NaP was measured every 20 s and plotted with open circles (left). The scale bar shows 20 μm RIL application. The current-voltage curve (right) from a mouse experiment is shown at three time points during the protocol. The mean respiratory period at RIL concentrations from 10-200 μm is plotted as a function of time from n = 3 rat slices tested at each concentration (no error bars).

Figure 4.

Dose-response curves for RIL. Closed circles show mean time to abolish XII activity obtained from rat experiments (n = 3 at each dose, no error bars), EC₅₀ = 32 μm. Open circles show attenuation of I_NaP inrats and mice. Some data points were originally reported in the study by Del Negro et al. (2002a).

Figure 6.

The effects of 20 nm TTX on respiratory rhythm in vitro. A, XII output is shown in control, 20 nm TTX (15 and 20 min elapsed time), and recovery conditions. B, Evoked action potentials in control and 20 nm TTX conditions. Baseline V_M was -60 mV. The calibration bar applies to both V_M and stimulus current (I_a). C, Bar charts showing the mean effects of TTX on cellular and systems level respiratory activity (n = 9 mouse slices tested). Frequency in TTX was taken as the average of three to five bursts just before TTX-induced cessation of the rhythm. The amplitude of XII motor output is scaled in arbitrary units (a.u.). Rheobase and spike “active fraction” are defined in Results. Error bars indicate SEM for n = 9 mouse slice preparations. Single asterisk indicates statistical significance at p < 0.01, and double asterisk indicates p < 0.005.

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.

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.

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).

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.