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. 2018 Aug;596(15):3245-3269.
doi: 10.1113/JP274727. Epub 2017 Jul 27.

Release of ATP by pre-Bötzinger complex astrocytes contributes to the hypoxic ventilatory response via a Ca2+ -dependent P2Y1 receptor mechanism

Vishaal Rajani  1   2 , Yong Zhang  1 , Venkatesh Jalubula  1 , Vladimir Rancic  1 , Shahriar SheikhBahaei  3   4 , Jennifer D Zwicker  1 , Silvia Pagliardini  1 , Clayton T Dickson  5 , Klaus Ballanyi  1 , Sergey Kasparov  6 , Alexander V Gourine  4 , Gregory D Funk  1
Affiliations

Release of ATP by pre-Bötzinger complex astrocytes contributes to the hypoxic ventilatory response via a Ca2+ -dependent P2Y1 receptor mechanism

Vishaal Rajani et al. J Physiol. 2018 Aug.

Abstract

Key points: The ventilatory response to reduced oxygen (hypoxia) is biphasic, comprising an initial increase in ventilation followed by a secondary depression. Our findings indicate that, during hypoxia, astrocytes in the pre-Bötzinger complex (preBötC), a critical site of inspiratory rhythm generation, release a gliotransmitter that acts via P2Y1 receptors to stimulate ventilation and reduce the secondary depression. In vitro analyses reveal that ATP excitation of the preBötC involves P2Y1 receptor-mediated release of Ca2+ from intracellular stores. By identifying a role for gliotransmission and the sites, P2 receptor subtype, and signalling mechanisms via which ATP modulates breathing during hypoxia, these data advance our understanding of the mechanisms underlying the hypoxic ventilatory response and highlight the significance of purinergic signalling and gliotransmission in homeostatic control. Clinically, these findings are relevant to conditions in which hypoxia and respiratory depression are implicated, including apnoea of prematurity, sleep disordered breathing and congestive heart failure.

Abstract: The hypoxic ventilatory response (HVR) is biphasic, consisting of a phase I increase in ventilation followed by a secondary depression (to a steady-state phase II) that can be life-threatening in premature infants who suffer from frequent apnoeas and respiratory depression. ATP released in the ventrolateral medulla oblongata during hypoxia attenuates the secondary depression. We explored a working hypothesis that vesicular release of ATP by astrocytes in the pre-Bötzinger Complex (preBötC) inspiratory rhythm-generating network acts via P2Y1 receptors to mediate this effect. Blockade of vesicular exocytosis in preBötC astrocytes bilaterally (using an adenoviral vector to specifically express tetanus toxin light chain in astrocytes) reduced the HVR in anaesthetized rats, indicating that exocytotic release of a gliotransmitter within the preBötC contributes to the hypoxia-induced increases in ventilation. Unilateral blockade of P2Y1 receptors in the preBötC via local antagonist injection enhanced the secondary respiratory depression, suggesting that a significant component of the phase II increase in ventilation is mediated by ATP acting at P2Y1 receptors. In vitro responses of the preBötC inspiratory network, preBötC inspiratory neurons and cultured preBötC glia to purinergic agents demonstrated that the P2Y1 receptor-mediated increase in fictive inspiratory frequency involves Ca2+ recruitment from intracellular stores leading to increases in intracellular Ca2+ ([Ca2+ ]i ) in inspiratory neurons and glia. These data suggest that ATP is released by preBötC astrocytes during hypoxia and acts via P2Y1 receptors on inspiratory neurons (and/or glia) to evoke Ca2+ release from intracellular stores and an increase in ventilation that counteracts the hypoxic respiratory depression.

Keywords: ATP; P2Y1; glia; gliotransmission; hypoxia; hypoxic ventilatory response; preBötzinger complex; purinoceptor.

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Figures

Figure 1
Figure 1. Purinergic modulation of respiratory rhythm: a working hypothesis
At the synapse of two preBötC inspiratory neurons, volleys of action potentials during inspiration in the presynaptic neuron evoke glutamate release, exciting postsynaptic neurons via ionotropic (GluR) and metabotropic (mGluR) receptors on preBötC inspiratory neurons. During hypoxia, astrocytes sense changes in O2, causing increases in intracellular calcium (i), resulting in the exocytotic release of gliotransmitters, including ATP (ii). ATP acts via P2Y1 receptors located on preBötC neurons (iii), causing a release of Ca2+ from intracellular stores mediated by DAG/IP3 (iv), and the modulation of downstream ion channels, [Ca2+]i, or the activation of protein kinase C (PKC) (v). Autocrine/paracrine of astrocytes by ATP may also contribute (vi).
Figure 2
Figure 2. Baseline parameters in the naïve control rats and rats transduced to express eGFP or TeLC in the preBötC astrocytes
Group data indicating the absolute values of respiratory frequency (A), mean arterial pressure (B), arterial PO2 and PCO2 (C), and pH (D) under baseline conditions, prior to the administration of hypoxia for the naïve control and viral control, as well as the TeLC expressing rats. An asterisk (*) indicates significant difference between means (P < 0.05, one‐way ANOVA, Tukey's post hoc test).
Figure 3
Figure 3. Expression of tetanus toxin light chain (TeLC) protein in medullary astrocytes attenuates the hypoxic ventilatory response
A, representative recordings obtained in naïve control and TeLC expressing rats showing changes in ∫PN, instantaneous frequency (inst. freq., breaths min–1) and blood pressure during 5.5 min of exposure to 10% O2. B, time course of relative frequency, ∫PNA and ventilatory output (frequency × ∫PNA) calculated for the three groups: naïve controls (n = 10), viral controls (n = 6) expressing eGFP in astroglia, and rats expressing eGFP and TeLC in astroglia (n = 6). Phases I and II of the HVR are shaded in grey. C, box plots comparing phase I and II parameters across the three groups. An asterisk (*) indicates a significant difference between means (P < 0.05, one‐way ANOVA, Tukey's post hoc test).
Figure 4
Figure 4. Viral expression was centered at the level of the preBötC and limited to astrocytes
A, schematic showing relative location of peak viral expression with respect to the preBötC in adult rats targeted with AVV‐sGFAP‐eGFP‐TeLC. Inferior olive dorsal (IOD), inferior olive medial (IOM), inferior olive principal (IOP), fourth ventricle (IV), nucleus ambiguus (NA), pyramidal tract (PY), rostral ventrolateral medulla (RVL), spinal trigeminal tract (Sp5), spinal trigeminal interpolar (Sp5I) and solitary tract (Sol). Viral expression was centered in the preBötC and dropped off substantially 500 μm rostral and caudal to the injection site that was centered in the preBötC. B, region expressing TeLC‐eGFP adenovirus is centered in the region containing a high density of NK1 receptor expressing neurons. C, double‐labelling of cells with the neuronal marker NeuN and the glial specific adenovirus, detected via eGFP, was not detected. Ten NeuN labelled cells are circled to facilitate comparison between images.
Figure 5
Figure 5. Functional identification of preBötC via microinjections of DLH
Unilateral injection of DLH (10 mm) into the VLM of urethane‐anaesthetized, paralysed adult rats when measuring instantaneous respiratory frequency (inst. freq., breaths min–1) from ∫PN. A, unilateral injection of DLH into the preBötC produces a robust increase in respiratory frequency. B, injection of DLH 200 μm rostral to the preBötC (presumably in the BötC) reduced the inspiratory frequency. C, DLH injection 300 μm caudal to the preBötC had minimal effect on inspiratory rhythm.
Figure 6
Figure 6. Activation of P2Y1 receptors in the preBötC of adult rat in vivo with MRS 2365 (1 mm) evokes an increase in respiratory frequency and a decrease in tidal volume (VT)
A, airflow and DIA EMG recordings show the response of an adult anaesthetized vagotomized rat to unilateral injection of MRS 2365 (1 mm, 200 nl) into the preBötC. B, time course of instantaneous frequency (inst. freq., breaths min–1), V T and minute ventilation (V˙E) responses evoked by a single injection of MRS 2365. C, group data (n = 8) illustrating the change in frequency, V T and V˙E relative to control (*significant different from predrug control values, P < 0.05, paired t test). D, schematic showing eight injection sites. Inferior olive dorsal (IOD), inferior olive medial (IOM), inferior olive principal (IOP), fourth ventricle (IV), nucleus ambiguous (NA), pyramidal tract (PY), rostral ventrolateral medulla (RVL), spinal trigeminal tract (Sp5), spinal trigeminal interpolar (Sp5I) and solitary tract (Sol).
Figure 7
Figure 7. Adenosine (500 μm) microinjected into the preBötC of adult rat in vivo has no effect on respiratory frequency or tidal volume (VT)
A, airflow, DIA and genioglossus (GG) EMG recordings show the response of an adult anaesthetized vagotomized rat to unilateral injection of adenosine into the preBötC. B, airflow, diaphragm (DIA) and genioglossus (GG) EMG recordings show the response of an adult anaesthetized vagotomized rat to the injection of DLH (1 mm) into the preBötC. C, group data illustrating the change in frequency and V T relative to control (n = 8) (*significant difference from predrug controls values, P < 0.05, paired t test). D, schematic showing eight injection sites in relation to inferior olive dorsal (IOD), inferior olive medial (IOM), inferior olive principal (IOP), fourth ventricle (IV), nucleus ambiguus (NA), pyramidal tract (PY), rostral ventrolateral medulla (RVL), spinal trigeminal tract (Sp5), spinal trigeminal interpolar (Sp5I) and solitary tract (Sol). E, tissue section showing injection site marked with fluorescent microspheres (yellow). PreBötC neurons (NK1+) are present in the region of the injection site.
Figure 8
Figure 8. Unilateral inhibition of P2Y1 receptors in the preBötC of adult paralysed rats in vivo with MRS 2279 increases the secondary hypoxic respiratory depression
A, representative traces showing changes in ∫PN and instantaneous frequency (inst. freq., breaths min–1) evoked by exposure to 5 min of 10% O2 following injection of vehicle (HEPES) or MRS 2279 into the preBötC. B, left: time courses of relative frequency, ∫PNA and ventilatory output (frequency × ∫PNA) calculated before and after injection of MRS 2279 (n = 6). Phases I and II of the HVR are shaded in grey. Right: comparisons of phase I and II parameters between vehicle and MRS 2279 responses [*significant difference between control (HEPES) and MRS 2279 trials, P < 0.5, paired t test]. C, schematic showing six injection sites. Inferior olive dorsal (IOD), inferior olive medial (IOM), inferior olive principal (IOP), fourth ventricle (IV), nucleus ambiguus (NA), pyramidal tract (PY), rostral ventrolateral medulla (RVL), spinal trigeminal tract (Sp5), spinal trigeminal interpolar (Sp5I) and solitary tract (Sol).
Figure 9
Figure 9. The hypoxic ventilatory response does not change with repeated exposure; time‐matched controls
A, representative traces from the same animal showing changes in ∫PN and instantaneous frequency (inst. freq., breaths min–1) during two consecutive hypoxic exposures (1 h apart) following stereotaxic injection of vehicle (Hepes) into the preBötC. B, left: time courses of relative frequency, ∫PNA and ventilatory output (frequency × ∫PNA) calculated before and after injection of MRS 2279 (n = 6). Phases I and II of the HVR are shaded in grey. Right: box plots comparing these same parameters during phases I and II of the HVR during consecutive exposures to hypoxia under control conditions (i.e. after Hepes injection).
Figure 10
Figure 10. Increases in [Ca2+]i contribute to the P2Y1 receptor mediated frequency increase in vitro
A, representative traces of integrated hypoglossal nerve output (∫XII) evoked in response to P2Y1 agonist MRS 2365 (10 s, 100 μm) before and after the local application of the cell permeable calcium chelator EGTA‐AM (1 mm) to the preBötC. B, box plot summarizing group data (*significant difference between means, n = 6; P = 0.0047, paired t test). C, representative traces ∫XII nerve output evoked in response to MRS 2365 (10 s, 100 μm) in control and after local application of thapsigargin (30 min, 200 μm). D, box plot of group data showing the effects of thapsigargin on the MRS 2365 evoked frequency increase (two left‐most boxes, n = 6) and time‐matched control experiments showing the effects of repeated vehicle injections (0.2% DMSO, n = 5) on the MRS 2365 response (* indicates significant difference between means, P < 0.05, paired t test).
Figure 11
Figure 11. Activation of P2Y1 receptors on preBötC inspiratory neurons in vitro evokes a thapsigargin‐sensitive, increase in [Ca2+]i
A, multiphoton images of baseline Fluo‐4 fluorescence indicating [Ca2+]i in five inspiratory neurons at a single optical plain (top), inspiratory‐related oscillations in [Ca2+]i and the peak [Ca2+]i increase evoked in response to locally applied MRS 2365 (100 μm, 10 s) (bottom) (B) [Ca2+]i increases in the neurons of (A) are synchronous with ∫XII and their responses to locally applied MRS 2365 (100 μm, 10 s). C, group data showing an increase of ∼50% in peak Fluo‐4 fluorescence in response to local application of MRS 2365 (100 μm, 10 s) over inspiratory preBötC neurons (n = 18, five slices; *significant difference between means, P < 0.0001, paired t test); AU, arbitrary units. D, multiphoton images of Fluo‐4 Ca2+ fluorescence in two inspiratory neurons at a single optical plain at baseline (top), at the peak of two control MRS 2365‐evoked (10 μm, 1 min, bath application) Ca2+ flourescence responses (top middle, bottom middle), seperated by 30 min intervals. The bottom image shows the response evoked by MRS 2365 after bath application of thapsigargin (100 μm, 30 min). E, traces of Fluo‐4 Ca2+ flourescence responses evoked by MRS 2365 in the same neurons shown in (D). F, box plot of group data comparing the decrease in peak MRS 2365‐evoked Fluo‐4 Ca2+ response that occurred between control responses 1 and 2 with the decrease in the MRS 2365‐evoked Ca2+ response that occurred between the second control MRS 2365 response and the MRS 2365 response in thapsigargin (or CPA) (n = 12, five slices; *significant difference between means, P < 0.0001, paired t test).
Figure 12
Figure 12. Responses of cultured preBötC glia to ATP are sensitive to depletion of intracellular calcium stores
A, images showing a phase contrast image of cultured glia (far left) and fluo‐4 Ca2+ fourescence under baseline conditions (middle‐left), during local application of ATP (10 μm, 10 s) (middle‐right) and during local application of ATP after pre‐application of SERCA inhibitor thapsigargin (50 nm, 30 min) (far right). B, time course of ATP‐evoked fluorescence changes measured from four regions of interest (numbered in A) in control (left) and after thapsigargin application (middle). Group data (n = 54 cells, from four culture plates, each plate was from one animal) showing relative changes in fluorescence (F peak/F baseline) in response to ATP and to ATP in the presence of thapsigargin (right) (n = 54 cells; *significant difference between means P < 0.001, paired t test).
Figure 13
Figure 13. Effect of CNQX and AP5/MK‐801 on ATP currents evoked in preBötC inspiratory neurons in TTX
A, representative traces showing an ATP current evoked in an inspiratory preBötC neuron in a rhythmic slice (note inspiratory synaptic currents pre‐TTX), after TTX and again in TTX after bath application of CNQX (10 μm) and AP5 (100 μm). B, group data comparing the relative reduction in the ATP current that occurs with consecutive applications in a time matched‐control group (n = 6 cells from six slices) with the reduction that was produced by CNQX+AP5/MK‐801 group (n = 8 from eight slices).
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