Modulation of hypoglossal motoneuron excitability by NK1 receptor activation in neonatal mice in vitro

Abstract

1. The effects of substance P (SP), acting at NK1 receptors, on the excitability and inspiratory activity of hypoglossal (XII) motoneurons (MNs) were investigated using rhythmically active medullary-slice preparations from neonatal mice (postnatal day 0-3). 2. Local application of the NK1 agonist [SAR(9),Met (O(2))(11)]-SP (SP(NK1)) produced a dose-dependent, spantide- (a non-specific NK receptor antagonist) and GR82334-(an NK1 antagonist) sensitive increase in inspiratory burst amplitude recorded from XII nerves. 3. Under current clamp, SP(NK1) significantly depolarized XII MNs, potentiated repetitive firing responses to injected currents and produced a leftward shift in the firing frequency-current relationships without affecting slope. 4. Under voltage clamp, SP(NK1) evoked an inward current and increased input resistance, but had no effect on inspiratory synaptic currents. SP(NK1) currents persisted in the presence of TTX, were GR82334 sensitive, were reduced with hyperpolarization and reversed near the expected E(K). 5. Effects of the alpha(1)-noradrenergic receptor agonist phenylephrine (PE) on repetitive firing behaviour were virtually identical to those of SP(NK1). Moreover, SP(NK1) currents were completely occluded by PE, suggesting that common intracellular pathways mediate the actions of NK1 and alpha(1)-noradrenergic receptors. In spite of the similar actions of SP(NK1) and PE on XII MN responses to somally injected current, alpha(1)-noradrenergic receptor activation potentiated inspiratory synaptic currents and was more than twice as effective in potentiating XII nerve inspiratory burst amplitude. 6. GR82334 reduced XII nerve inspiratory burst amplitude and generated a small outward current in XII MNs. These observations, together with the first immunohistochemical evidence in the newborn for SP immunopositive terminals in the vicinity of SP(NK1)-sensitive inspiratory XII MNs, support the endogenous modulation of XII MN excitability by SP. 7. In contrast to phrenic MNs (Ptak et al. 2000), blocking NMDA receptors with AP5 had no effect on the modulation of XII nerve activity by SP(NK1). 8. In conclusion, SP(NK1) modulates XII motoneuron responses to inspiratory drive primarily through inhibition of a resting, postsynaptic K+ leak conductance. The results establish the functional significance of SP in controlling upper airway tone during early postnatal life and indicate differential modulation of motoneurons controlling airway and pump muscles by SP.

Figure 1. Schematic of transverse medullary slice preparation

The inspiratory related rhythm is generated in the pre-Bötzinger complex. Rhythmic bursts of activity are transmitted to XII MNs (curved arrow) and along the MN axons into the XII nerves. The diagram illustrates the arrangement of suction electrodes for recording inspiratory activity from the XII nerve (nerve activity is typically recorded bilaterally), pressure ejection pipette for local application of drugs and whole-cell recording pipettes for recording membrane voltage or current from individual MNs under current or voltage clamp, respectively. Examples of XII MN membrane potential (V_m), membrane current (I_m) and integrated XII nerve activity (∫XII) during inspiration are also shown. NA, nucleus ambiguus; IO, inferior olive; 5 SP, spinal trigeminal nucleus; XII, hypoglossal nucleus; pre-BötC, pre-Bötzinger complex.

Figure 2. SP_NK1 causes tonic excitation of XII nerve output and potentiates inspiratory burst amplitude

Long time course, recordings of integrated left and right XII nerve activity from a single rhythmic slice (P3) showing response to 10 s unilateral application over the hypoglossal nucleus of 0.01 (A), 0.1 (B) and 1.0 μm SP_NK1 (C). Effects of SP_NK1 on burst profile are shown to the right of each panel as the average of 10 cycles taken before (control), during (0.01 μm SP_NK1) and after (washout) application of SP_NK1.

Figure 3. SP_NK1 potentiation of inspiratory activity is dose dependent

A, time course of changes in integrated inspiratory burst amplitude (mean ±s.e.m.) following 30 s applications of 0.01, 0.1 and 1.0 μm SP_NK1 over the XII nucleus (agonist applied starting at time t = 0; n = 9). B, dose-dependent effects of SP_NK1 on inspiratory XII nerve burst amplitude (n = 9).

Figure 4. SP_NK1 potentiation of XII nerve inspiratory burst amplitude is reduced by neurokinin receptor antagonists

A, effects of 0.1 μm SP_NK1 locally applied for 10 s on integrated XII nerve activity before (control) and after 1 and 3 h of incubation with spantide (5 μm). B, effects 0.1 μm SP_NK1 locally applied for 10 s before (control) during and 15 min after (washout) a 5 min pre-application of 10 μm GR-82334 locally over the XII nucleus. C, mean (±s.e.m.) inhibition by spantide (5 μm, bath applied, n = 5) or GR82334 (10 μm, locally applied, n = 8) of the SP_NK1-mediated potentiation of inspiratory burst amplitude (0.1 μm SP_NK1, 10 s local application). * Significant increase from control. Note, spantide and GR82334 were tested in different preparations.

Figure 5. SP_NK1 has no effect on the magnitude of inspiratory synaptic currents

A, long time course voltage-clamp recording of a XII MN showing effect on membrane current (I_m) of locally applying 1.0 μm SP_NK1 over the XII nucleus. Peaks represent individual inspiratory synaptic currents (* series resistance tests or pulse protocols to establish input resistance). B, no effect of SP_NK1 on the inspiratory synaptic current envelope is shown for a single XII MN as the average of 10 cycles before, during and after local application of SP_NK1. C, average time course of relative changes in peak amplitude and charge transfer of inspiratory synaptic currents following a 10 s local application of 1.0 μm SP_NK1 (starting time at t = 0) over the XII nucleus (n = 11).

Figure 6. Potentiation of inspiratory XII activity is not dependent on NMDA receptor activation

Potentiation of inspiratory burst amplitude by 30 s local application of 0.1 μm SP_NK1 was the same in control and after a 2 min local pre-application of 1 mm AP5 (n = 3).

Figure 7. SP_NK1 depolarizes XII MNs and increases MN excitability

A, long time scale current-clamp recording of XII MN membrane voltage (V_m) showing the response to a sustained local application of 0.1 μm SP_NK1 and a repetitive firing protocol. Note that DC injection was employed during the SP_NK1 application to counter the SP_NK1-induced depolarization. Lower case letters in A indicate points at which traces with higher temporal resolution shown in B and C occurred. B, firing responses of the MN in A to individual inspiratory synaptic currents during control conditions (a), during SP_NK1-mediated depolarization (c), during continued application of SP_NK1 but following DC injection (d), and during recovery (g). C, repetitive firing responses to square-wave current pulses injected during control (b), during continued SP_NK1 but after DC injection (e), and following washout (f) (spikes are truncated). D, firing frequency-current relationships for a single XII MN before (control), during and after (washout) local application of 1.0 μm SP_NK1.

Figure 8. SP_NK1 currents persist in TTX and are sensitive to GR82334

A, membrane current induced by 1 μm SP_NK1 under control conditions, following a 2 min pre-application of GR82334 and after 15 min of washout, all in the presence of 1 μm tetrodotoxin (TTX). Note the small outward current induced by the application of GR82334 (* series resistance tests or pulse protocols to establish input resistance). B, current-voltage relationship for the SP_NK1 current induced before and after 2 min pre-application of 100 μm GR82334.

Figure 9. The reversal potential and magnitude of the SP_NK1 current are sensitive to extracellular [K⁺]

A, slow time scale recordings showing effects on the SP_NK1 current of changing extracellular [K⁺] from 9 to 3 mm. B, whole-cell current-voltage plots, obtained in the presence of 1.0 μm TTX, showing the effects of changing extracellular [K⁺] on membrane current and the SP_NK1-induced current (SP-control). C, the current-voltage relationships of the SP_NK1 currents shown in B are enlarged to illustrate the effects of changing extracellular [K⁺].

Figure 10. SP_NK1 and the α₁-noradrenergic receptor agonist PE modulate excitability through similar postsynaptic pathways

A, long time scale recording of membrane current responses to 90 s local application of 20 μm PE (left trace), 60 s local application of 1.0 μm SP_NK1 15 min later (middle trace) and the co-application of PE and SP_NK1 a further 15 min later (right trace). B, whole-cell current-voltage relationships obtained under control, during the application of PE alone, during co-application of PE and SP_NK1, and during washout. C, the current-voltage relationships of the current produced by PE alone (PE − control) and in combination with SP ((PE + SP_NK1) − control).

Figure 11. SP_NK1 and PE have similar effects on the repetitive firing behaviour of XII MNs

Firing frequency-current relationships for a XII MN, elicited by somal injection of square-wave current pulses immediately before and during local application of SP_NK1 (1.0 μm) or PE (10 μm). Response shown is typical of all cells tested (SP_NK1, n = 8; PE, n = 3).

Figure 12. SP_NK1 and PE differentially modulate inspiratory activity

A, long time course recordings of integrated XII nerve activity from rhythmic slices (P3) showing response to 10 s unilateral application over the hypoglossal nucleus of 1.0 μm SP_NK1 or 10 μm PE. B, dose-dependent effects of SP_NK1(n = 9, from Fig. 3B) and PE (n = 6) on inspiratory XII nerve burst amplitude. C, effect of PE (10 μm) on the inspiratory synaptic current envelope is shown for a single XII MN as the average of 10 cycles before and during local application of PE. Bar chart indicates effects of PE on relative peak inspiratory current and charge transferred per inspiratory cycle averaged for 8 XII MNs.

Figure 13. XII nerve inspiratory burst amplitude is modulated by an endogenously released neurokinin

A, XII inspiratory burst profiles averaged in a single slice from 10 inspiratory cycles before (control), during and after (washout) 5 min local application of 10 μm GR82334. B, average effects of locally applying GR82234 (10 μm, 5 min) on relative XII nerve inspiratory burst amplitude (n = 8). * Significant difference from control.

Figure 14. Immunolocalization of SP-containing terminals in the XII nucleus

A, photomicrograph of a cross-section through the medulla of a P2 mouse at the level of the XII nucleus showing substance P-like immunoreactive terminals in the XII nucleus. B, high magnification view of a region within the ventromedial aspect of the XII nucleus showing punctate substance P immunolabelling (small arrows) in close proximity to biocytin-labelled dendrite (large arrowhead) of a SP_NK1-sensitive inspiratory XII MN. Abbreviations: XII, XII nucleus; CC, central canal; DMV, dorsal motor vagus.