Postnatal development and activation of L-type Ca2+ currents in locus ceruleus neurons: implications for a role for Ca2+ in central chemosensitivity

Abstract

Little is known about the role of Ca(2+) in central chemosensitive signaling. We use electrophysiology to examine the chemosensitive responses of tetrodotoxin (TTX)-insensitive oscillations and spikes in neurons of the locus ceruleus (LC), a chemosensitive region involved in respiratory control. We show that both TTX-insensitive spikes and oscillations in LC neurons are sensitive to L-type Ca(2+) channel inhibition and are activated by increased CO(2)/H(+). Spikes appear to arise from L-type Ca(2+) channels on the soma whereas oscillations arise from L-type Ca(2+) channels that are distal to the soma. In HEPES-buffered solution (nominal absence of CO(2)/HCO(3)(-)), acidification does not activate either oscillations or spikes. When CO(2) is increased while extracellular pH is held constant by elevated HCO(3)(-), both oscillation and spike frequency increase. Furthermore, plots of both oscillation and spike frequency vs. intracellular [HCO(3)(-)]show a strong linear correlation. Increased frequency of TTX-insensitive spikes is associated with increases in intracellular Ca(2+) concentrations. Finally, both the appearance and frequency of TTX-insensitive spikes and oscillations increase over postnatal ages day 3-16. Our data suggest that 1) L-type Ca(2+) currents in LC neurons arise from channel populations that reside in different regions of the neuron, 2) these L-type Ca(2+) currents undergo significant postnatal development, and 3) the activity of these L-type Ca(2+) currents is activated by increased CO(2) through a HCO(3)(-)-dependent mechanism. Thus the activity of L-type Ca(2+) channels is likely to play a role in the chemosensitive response of LC neurons and may underlie significant changes in LC neuron chemosensitivity during neonatal development.

Fig. 1.

A: typical appearance of both tetrodotoxin (TTX)-insensitive oscillations and spikes [from a postnatal day 10 (P10) animal]. Arrow marks injection of depolarizing current to induce spikes. B, top: addition of nifedipine results in the complete elimination of both oscillations and spikes. Addition of depolarizing current (arrow) in the presence of nifedipine fails to elicit spikes. B, bottom: washing the nifedipine-exposed neuron with normocapnic aCSF + TTX restores the appearance of both oscillations and spikes. C: summary of the effects of nifedipine on oscillations and spikes. Note that nifedipine completely abolishes both TTX-insensitive oscillations and spikes. Bars represent means ± SE. n = 5.

Fig. 2.

Effects of membrane potential on TTX-insensitive spikes and oscillations. A: arrows mark the injection of either hyperpolarizing or depolarizing current into the soma through the whole cell patch pipette. Small hyperpolarizing or depolarizing injections (sufficient for a <5-mV change in membrane potential) result in either decreases or increases in spike frequency, respectively. B: arrows mark the injection of hyperpolarizing current into TTX-exposed locus ceruleus (LC) neurons. Large hyperpolarizing current injections cause no change in either oscillation amplitude or frequency. C: addition of 100 μM carbenoxolone (in 5% CO₂) inhibits TTX-insensitive oscillations (right) but does not affect TTX-insensitive spikes (left).

Fig. 3.

Simultaneous whole cell patch and Fura-2 imaging, showing TTX-insensitive spikes (A, C) or oscillations (B) plus changes in intracellular (somal) Ca²⁺. A : increase in spike frequency causes a concurrent increase in somal Ca²⁺ levels. Arrow marks the injection of hyperpolarizing current that eliminates spikes and results in a return to baseline Ca²⁺ levels. B: presence or absence of TTX-insensitive oscillations has no effect on soma Ca²⁺ levels. C: large (>0.1 R_fl) and small (<0.1 R_fl) relative increases in intracellular Ca²⁺ levels compared with the corresponding increases in spike frequency. Larger changes in spike frequency are significantly correlated with larger changes in somal Ca²⁺. Bars represent means ± SE.

Fig. 4.

Effects of the removal of CO₂/HCO₃⁻ on TTX-insensitive oscillations and spikes (from a P12 rat). A: variable, typically absent appearance of oscillations in HEPES-buffered aCSF equilibrated with 100% O₂. Inset: a large injection of depolarizing current is necessary to induce TTX-insensitive spikes. B: changing from HEPES solution to normocapnic, HCO₃⁻-buffered aCSF results in the restoration of both TTX-insensitive oscillations and spikes. Notice the appearance of spikes without the addition of depolarizing current. C: returning to HEPES-buffered aCSF solution once again results in the inhibition of both spikes and oscillations.

Fig. 5.

Reversible increases in TTX-insensitive spike (A) and oscillation (B) frequency due to hypercapnia (15% CO₂), indicating that these spikes and oscillations can be stimulated by increased CO₂/H⁺.

Fig. 6.

Simultaneous whole cell patch and loading with a pH-sensitive dye, showing pH_i changes and the effects on the TTX-insensitive current. A: hypercapnic acidosis (HA) causes a large decrease in pH_i, whereas isohydric hypercapnia (IH) causes a smaller, more variable decrease in pH_i. B: HA and IH result in similar frequency and amplitude for both oscillations and spikes despite their different effects on pH_i. C: CO₂ causes dose-dependent increases in the TTX-insensitive spike frequency. Despite the diminished intracellular and extracellular acidification seen with IH solutions, no decrease in spike rate is observed in 15% CO₂ (HA vs. IH). HA and IH ΔHz values (firing rate in 15% CO₂ − firing rate in 5% CO₂) are significantly increased from ΔHz values for 10% CO₂ (firing rate in 10% CO₂ − firing rate in 5% CO₂), with P < 0.01 and P < 0.001, respectively. Bars represent mean ± SE.

Fig. 7.

A: plot of intracellular HCO₃⁻ (mM) vs. TTX-insensitive spike frequency. Values were taken in neurons with membrane potentials between −32 to −37 mV. B: plot of intracellular HCO₃⁻ (mM) vs. TTX-insensitive oscillation frequency. In this chart, a clear age-related development of oscillation frequency is observed.

Fig. 8.

An age-related development in TTX-insensitive spike frequency is observed similar to that noted for TTX-insensitive oscillations (Fig. 7B). All values were taken without the addition of depolarizing current in normocapnic aCSF. Age group values were significantly different from one another with P < 0.001. >P13: n = 9 from 5 neurons, 5 slices; P12–P10: n = 36 from 24 neurons, 16 slices; P7–P9: n = 5 from 4 neurons, 4 slices. Bars represent means ± SE.

Fig. 9.

Observations of TTX-insensitive spikes and oscillations in LC neurons from rats ages P3–P10. Prior to age ∼P9, a transition period exists whereby oscillations and spikes can only be observed in hypercapnic aCSF. A: record from a P8 animal showing activation of both TTX-insensitive spikes and oscillations in 15% CO₂. When 5% CO₂ is restored, both spikes and oscillations are no longer present. B: a comparison of the appearance and amplitude of TTX-insensitive oscillations/spikes activated by CO₂ vs. age of neonatal rat. All records were taken in hypercapnia (15% CO₂). Note that in a young neonate (P3), even in 15% CO₂, no oscillations or spikes are seen. In neonates aged P5–P7, oscillations but not spikes are apparent in 15% CO₂. In a neonate aged P10, spikes are clearly evident in 15% CO₂.

Fig. 10.

Summary of the effects of age on the appearance of TTX-insensitive spikes (A) and oscillations (B). In these records, depolarizing current was injected to bring membrane potential to −20 mV. Any spikes observed under these conditions in either 15% CO₂ or 5% and 15% CO₂ were recorded.

Fig. 11.

Model of the chemosensitive K⁺- and Ca²⁺-sensitive pathways in LC neurons. Numbered pathways are referred to in the text. Left represents a summary of the proposed role of H⁺-sensitive K⁺ channels in the hypercapnic depolarization and increase in firing rate in chemosensitive LC neurons. Right depicts a possible pathway for hypercapnic activation of Ca²⁺ channels and potential roles for Ca²⁺ in chemosensitive signaling in LC neurons.