Metabotropic glutamate receptors (mGluR5) activate transient receptor potential canonical channels to improve the regularity of the respiratory rhythm generated by the pre-Bötzinger complex in mice

Abstract

Metabotropic glutamate receptors (mGluRs) are hypothesized to play a key role in generating the central respiratory rhythm and other rhythmic activities driven by central pattern generators (e.g. locomotion). However, the functional role of mGluRs in rhythmic respiratory activity and many motor patterns is very poorly understood. Here, we used mouse respiratory brain-slice preparations containing the pre-Bötzinger complex (pre-BötC) to identify the role of group I mGluRs (mGluR1 and mGluR5) in respiratory rhythm generation. We found that activation of mGluR1/5 is not required for the pre-BötC to generate a respiratory rhythm. However, our data suggest that mGluR1 and mGluR5 differentially modulate the respiratory rhythm. Blocking endogenous mGluR5 activity with 2-Methyl-6-(phenylethynyl)pyridine (MPEP) decreases the inspiratory burst duration, burst area and frequency, whereas it increases the irregularity of the fictive eupneic inspiratory rhythm generated by the pre-BötC. In contrast, blocking mGluR1 reduces the frequency. Moreover, the mGluR1/5 agonist 3,5-dihydroxyphenylglycine increases the frequency and decreases the irregularity of the respiratory rhythm. Based on previous studies, we hypothesized that mGluR signaling decreases the irregularity of the respiratory rhythm by activating transient receptor potential canonical (TRPC) channels, which carry a non-specific cation current (ICAN). Indeed, 3,5-dihydroxyphenylglycine (DHPG) application reduces cycle-by-cycle variability and subsequent application of the TRPC channel blocker 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl]imidazole (SKF-96365) hydrochloride reverses this effect. Our data suggest that mGluR5 activation of ICAN-carrying TRPC channels plays an important role in governing the cycle-by-cycle variability of the respiratory rhythm. These data suggest that modulation of TRPC channels may correct irregular respiratory rhythms in some central neuronal diseases.

Fig. 1. Mouse medullary slice preparations containing the neural network for inspiratory rhythm generation, the pre-Botzinger Complex

A) Transverse brainstem slice preparation with the ventral respiratory group (VRG). B) Raw VRG population activity (VRG) is recorded with an extracellular electrode, placed on the surface of the slice (bottom trace) and integrated VRG activity (∫VRG) (top trace). Integrated ∫VRG population inspiratory bursting is shown on a longer time scale. C1) The rhythmic VRG-island preparation containing the pre-BötC was isolated from the respiratory brainstem slice preparation. Rhythmic ∫VRG population activity was recorded from the VRG-island after its isolation from the rest of the brainstem slice (bottom trace). C2) Western blots verified the expression of mGluR5 and mGluR1 in the VRG-island, containing the pre-BötC. Cortex and cerebellum tissues served as positive controls for group 1 mGluR expression and GADPH was used as a loading control.

Fig. 2. The group I mGluR (1/5) agonist, DHPG improves the regularity of fictive eupnea and this effect can be reversed by TRPC antagonist, SKF-96365

Integrated VRG activity (∫VRG) with fictive inspiratory bursting, A1) during fictive eupneic activity under control conditions, in artificial cerebral spinal fluid (ACSF); A2) after bath-application of the group I mGluR (1/5) agonist, DHPG; and B1) subsequent co-application of the TRPC channel antagonist, SKF-96365. A3) Following bath-application of DHPG, fictive eupneic inspiratory rhythmic bursts become more regular (less irregular), shorter in duration and more frequent (*P < 0.05; **P < 0.01, paired t-test). B2) Subsequent co-application of the TRPC channel antagonist SKF-96365 reverses the DHPG improvement in rhythm regularity, without significantly changing the burst frequency. Statistical tests were made on raw data. C) After blocking TRPC channels with SKF-96365, subsequent additional blockade of INaP eliminated the inspiratory rhythm.

Fig. 3. Blocking group I mGluR (1 and 5) does not eliminate the inspiratory rhythm, but increases irregular rhythmic bursting and decreases the burst frequency

Integrated VRG activity (∫VRG) with fictive inspiratory bursting, A1) during fictive eupneic activity under control conditions, in artificial cerebral spinal fluid (ACSF), A2) after blockade of mGluR5 activation with MPEP and after A3) additionally blocking mGluR1 with LY367385. A4) In the presence of MPEP and LY36785, the inspiratory rhythm continues, but becomes more irregular, and lower in frequency, relative to that in control (ACSF) conditions (P* < 0.05, paired t-test). B) After blocking mGluR1 and mGluR5, subsequent bath application of the persistent sodium current blocker, riluzole eliminates the rhythm. Statistical tests were made on raw data.

Fig. 4. Blocking endogenous activation of mGluR 5 with MPEP results in shorter, less frequent and more irregular fictive eupneic inspiratory bursting

Integrated VRG activity (∫VRG) with fictive inspiratory bursting, A1) during fictive eupneic activity, under control conditions, in artificial cerebral spinal fluid (ACSF); and, A2) after blocking endogenous mGluR5 with MPEP. A3) In the presence of MPEP, the inspiratory rhythm becomes more irregular and the inspiratory burst area, burst duration and frequency are reduced relative to that recorded in control (ACSF) conditions (*P < 0.05; ***P < 0.001, paired t-test). A4) After blocking mGluR5 with MPEP, subsequent bath-application of the persistent sodium current (INaP) antagonist, riluzole eliminated the inspiratory rhythm. Statistical tests were made on raw data.

Fig. 5. Superfusion of MPEP into the pre-BötC region increases inspiratory burst irregularity, decreases burst area, duration and frequency

A) Schematic illustration of unilateral superfusion of MPEP or ACSF (vehicle control) into the pre-BötC, while recordingintegrated VRG activity (∫VRG) with fictive inspiratory bursting. A1) Fictive eupneic activity under control conditions, in artificial cerebral spinal fluid (ACSF); and, A2) after blocking endogenous mGluR5 with MPEP. A3) In the presence of MPEP, the inspiratory rhythm becomes more irregular and the inspiratory burst area, burst duration and frequency are reduced relative to that recorded in control (ACSF) conditions (*P < 0.05, paired t-test). Integrated VRG activity (∫VRG) with fictive inspiratory bursting is shown, B1) during fictive eupneic activity under control conditions in artificial cerebral spinal fluid (ACSF); and, B2) after superfusion of ACSF (vehicle control). B3) In contrast to MPEP, vehicle control injections of ACSF did not significantly alter inspiratory burst regularity, burst area, duration or frequency. Statistical tests were made on raw data.

Fig. 6. Blocking endogenous activation of mGluR 1 with LY367385 results a reduction in the frequency of fictive eupneic inspiratory bursting

Integrated VRG activity (∫VRG) with inspiratory bursting, A1) during fictive eupneic activity, under control conditions, in artificial cerebral spinal fluid (ACSF); and, A2) after blocking endogenous mGluR1 with LY367385. B) In the presence of LY367385, the inspiratory rhythm frequency is reduced relative to that recorded in control (ACSF) conditions (**P<0.01, paired t-test). Statistical tests were made on raw data.

Fig. 7. Blocking mGluR5 with MPEP increases inspiratory neuron burst irregularity and decreases neuron burst frequency while additionally blocking mGluR1 with LY367385 does not further alter these parameters

Fictive eupneic bursts (∫VRG) and inspiratory rhythmic neuron activity recorded in A1) control conditions (ACSF) and, A2) after blocking mGluR5 with MPEP. A3) Blocking mGluR5 with 20μM MPEP increases the inspiratory bursting irregularity, decreases inspiratory burst area and burst frequency without altering the inspiratory bursting duration. A4) Subsequent blockade of mGluR1 with LY367385 does not further alter A5) the bursting regularity, burst area, burst duration or burst frequency (*P < 0.05; **P < 0.01, paired t-test). B) The subsequent blockade of the persistent sodium current (INaP) with riluzole abolished the respiratory rhythm activity. Statistical tests were made on raw data.