Substance P modulation of TRPC3/7 channels improves respiratory rhythm regularity and ICAN-dependent pacemaker activity

Abstract

Neuromodulators, such as substance P (SubP), play an important role in modulating many rhythmic activities driven by central pattern generators (e.g. locomotion, respiration). However, the mechanism by which SubP enhances breathing regularity has not been determined. Here, we used mouse brainstem slices containing the pre-Bötzinger complex to demonstrate, for the first time, that SubP activates transient receptor protein canonical (TRPC) channels to enhance respiratory rhythm regularity. Moreover, SubP enhancement of network regularity is accomplished via selective enhancement of ICAN (inward non-specific cation current)-dependent intrinsic bursting properties. In contrast to INaP (persistent sodium current)-dependent pacemakers, ICAN-dependent pacemaker bursting activity is TRPC-dependent. Western Blots reveal TRPC3 and TRPC7 channels are expressed in rhythmically active ventral respiratory group island preparations. Taken together, these data suggest that SubP-mediated activation of TRPC3/7 channels underlies rhythmic ICAN-dependent pacemaker activity and enhances the regularity of respiratory rhythm activity.

Figure 1. Mouse medullary slice preparation containing the neural network for respiratory activity generation

A) Extracellular electrodes were placed on the surface of the VRG to record population activity. Intracellular electrodes were placed close to the extracellular electrodes to simultaneously record inspiratory neuron activity. B) Raw VRG population activity was integrated (ʃ); the integrated VRG activity is dominated by inspiratory neurons, giving rise to fictive inspiratory bursts in the integrated traces (ʃVRG). Lower trace is a simultaneous intracellular recording of inspiratory neuron bursting in phase with the population.

Figure 2. Substance P enhances the fictive eupneic rhythm generated by the ventral respiratory group (ʃVRG)

A1) Fictive eupnea (ʃVRG) recorded in control condition (ACSF) and after A2) bath application of 0.1 μM SubP. B) SubP decreases the fictive eupneic, ʃVRG burst irregularity while it enhances the burst area and increases the burst frequency (*, p<0.05 and *** p<0.001, paired t-test), without altering burst duration (statistical tests were made on raw data).

Figure 3. Substance P enhances respiratory rhythm via canonical transient receptor protein (TRPC) channels

A1) Fictive eupneic activity (ʃVRG) recorded in control condition (ACSF) and after A2) bath application of 0.1 μM SubP. B1) In the presence of SubP, subsequent bath application of 5-10μM SKF-96365, suppresses the SubP enhancement of VRG rhythm by SubP within ~10-15mins). B2) In the presence of SubP, subsequent bath application of SKF-96365 increases the burst irregularity and decreases the burst area, the burst duration and the rhythm frequency (*, p<0.05; **, p<0.01, paired t-test). C) In the presence of SubP and SKF-96365, additionally blocking the persistent sodium current (INaP) with 10μM of riluzole abolished the VRG respiratory rhythm. D) Following 20mins of SubP bath- application, subsequent co-application of SKF-96365 increased ʃVRG bursting irregularity, while burst area and frequency were similar to control values (ACSF), burst duration was reduced (**, p<0.01, paired t-test) (statistical tests were made on raw data).

Figure 4. Flufenamic acid reverses the improvement of the respiratory rhythm by the Substance P

A1) Fictive eupneic activity (ʃVRG) recorded in control (ACSF); and A2) following bath application of 0.1 μM SP for 20mins. B1) In the presence of SubP, subsequent bath application of 50μM of flufenamic acid (FFA) B2) reverses the SubP respiratory rhythm enhancement by increasing the rhythm irregularity and decreasing the burst area, the burst duration and the rhythm frequency (*, p<0.05; **, p<0.01; ***, p<0.001 paired t-test). C) In the presence of SubP and SKF-96365, subsequent additional application of 10μM riluzole completely abolished the ʃVRG rhythm. D) Following SubP bath- application for 20 mins., co-application of FFA, the ʃVRG bursting irregularity, burst area and frequency were similar to control values (ACSF only), while burst duration was reduced (***, p<0.001 paired t-test) (statistical tests were made on raw data).

Figure 5. Flufenamic acid prevents modulation of fictive eupnea (ʃVRG) via Substance P

A1) Fictive eupneic activity (ʃVRG) recorded in control condition (ACSF) and after A2) bath-application of FFA (50μM for 30 mins). B1) Pretreatment with FFA prevented the SubP enhancement of the fictive eupnea. B2) In the presence of FFA, subsequent bath co-application of SubP fails in improve the regularity, enhance the burst area and prolong the burst duration. However, an increase of the frequency (*, p<0.05; paired t-test) is observed. C) During FFA and SubP co-application, additional application of 10μM riluzole completely abolished the ʃVRG rhythm. D) After FFA-preincubation, rather than enhancing respiratory activity, subsequent SubP application resulted in ʃVRG rhythmic activity being more irregular, with a reduced burst area, while burst duration and frequency were similar, relative to control values (ACSF only) (*, p<0.05; **, p<0.01, paired t-test) (statistical tests were made on raw data).

Figure 6. The enhancement of respiratory rhythm regularity by Substance P does not involve the persistent sodium current (INaP)

A1) Fictive eupneic activity (ʃVRG) recorded in control conditions (ACSF) and after A2) bath-application of SubP (20 mins). B1) In the presence of SubP, subsequent bath application of 10μM of riluzole for 10mins. B2) does not affect the SubP-mediated decrease in fictive eupnea irregularity and enhanced burst area (see also Fig. 3). Only the burst duration and the frequency are decreased (*, p<0.05; **, p<0.01, paired t-test). C) In the presence of SubP and riluzole, subsequently adding 10μM of SKF96365 disrupts, then abolishes, the rhythm. D) In the presence of SubP and riluzole (Fig. 7B1), ʃVRG rhythmic activity is less irregular and has a higher frequency, while burst area and burst duration were similar to control values (ACSF only, Fig. 7A1, *, p<0.05; **, p<0.01, paired t-test) (statistical tests were made on raw data).

Figure 7. Calcium-dependant cation current (ICAN) is carried by TRPC3/7 channels

A1) ICAN pacemaker neuron activity recorded while bath-applying a mixture of synaptic antagonists, CNQX, CPP, bicuculine, strychnine. A2) SubP (0.1μM) enhances ICAN pacemaker bursting properties (green/red overlay) (Pena and Ramirez 2002). A3) Following 20mins. bath application of SubP, subsequent co-application of SKF96365 (5μM), an antagonist of canonical transient receptor protein (TRPC) channels, reverses the SubP-enhanced bursting properties, within 5 mins. (red/black overlay). A4), A longer term (~15min) perfusion SKF-96365 abolishes ICAN pacemaker bursting properties. After SKF-96365 application, depolarizing current injection did not trigger bursting activity (30pA steps, 2sec duration). B) After 20mins in SubP, subsequent co-application of SKF-96365, for 5mins. initially leads to an increase in the burst irregularity, decrease in the burst area, while the burst duration is initially the same as in SubP, while burst frequency is increased prior to reducing then eliminating bursting (*, p<0.05; ***, p<0.001, paired t-test) (statistical tests were made on raw data).

Figure 8. Persistent sodium current (INaP) pacemaking is not TRPC channel dependent

A1) INaP-dependent pacemaker neuron activity recorded while bath-applying a mixture of CNQX, CPP, bicuculine, strychnine. A2) Bath application of SubP (0.1μM, 20mins.) enhances INaP)-dependent pacemaker activity bursting properties (green/red overlay). A3) In the presence of SubP, subsequent bath application of SKF-96365 (5μM) 15mins),, does not block SubP-enhanced rhythmic bursting of INaP-dependent pacemakers (red/black overlay). A4) As expected, INaP-dependent pacemaker bursting properties are abolished within 10mins. following additionally applying riluzole 10μM. B) In the presence of SubP, the burst irregularity, the burst area, the burst duration or the frequency were not affected by subsequently adding SKF-96365 (statistical tests were made on raw data).

Figure 9. SubP-enhancement of inspiratory non-pacemaker activity is reversed by SKF-96365

A1) Fictive eupneic activity (ʃVRG) and inspiratory non-pacemaker neuron recorded in control conditions (ACSF) and after A2) bath-application of SubP (20 mins). A3) In the presence of SubP. subsequent bath co-application of SKF-96365 does not eliminate ʃVRG and inspiratory non-pacemaker bursting properties, while A4) subsequent additional co-application of riluzole eliminates inspiratory non-pacemaker rhythmic bursting. B1) SubP application decreases inspiratory non-pacemaker rhythm irregularity and increases burst frequency but did not significantly alter burst area or burst duration (*, p<0.05, paired t-test). B2) After 20mins. in SubP, subsequent co-application of SKF-96365 increased bursting irregularity, decreased burst area underlying action potentials and decreased burst frequency, without altering burst duration of inspiratory non-pacemakers (*, p<0.05, paired t-test). B3) Following 20mins of SubP bath-application, subsequent co-application of SKF-96365, non-pacemaker bursting irregularity, burst area, burst duration and burst frequency were similar to control values measured in ACSF alone (statistical tests were made on raw data).

Figure 10. Qualitative Western blots reveal the expression of canonical transient receptor proteins 3 and 7 (TRPC3 and TRPC7) in the VRG

A) ʃVRG activity recorded from the whole medullary slice, in control conditions (ACSF). B) ʃVRG activity recorded from the VRG-island after its isolation from the rest of the medullary slice, in control conditions (ACSF). C) NK1 receptors (10μg total protein/ lanes 1-3); and, D) TRPC1 channels are expressed in the cortex, but not in the cerebellum or VRG-island preparations, 15μg total protein/ lanes). TRPC3 channels are expressed in all the VRG-islands tested (15μg total protein/ lanes 1-3). TRPC4 channels are expressed in the cortex and cerebellum but not the VRG-islands, (15μg total protein/ lanes), whereas TRPC5 channels (15μg total protein/ lanes) and TRPC6 channels preparations, 15μg total protein/ lanes) are expressed in the cortex and, but not in the cerebellum or VRG-islands. TRPC7 channels are expressed in the cerebellum and VRG-islands (15μg total protein/ lanes). E) TRPM4 channels were detected in the cortex (10μg total protein/ lanes 1 and 2) and cerebellum (10μg total protein/ lanes 1 and 2) but does not appear to be expressed in the VRG-Island (VRG-Island preparations; 10μg total protein/ lanes 1 and 3; 50μg total protein/lanes 2 and 4).