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. 2017 Oct 10;8(1):838.
doi: 10.1038/s41467-017-00960-3.

D-serine released by astrocytes in brainstem regulates breathing response to CO2 levels

S Beltrán-Castillo  1 , M J Olivares  1 , R A Contreras  1 , G Zúñiga  1 , I Llona  1 , R von Bernhardi  2 , J L Eugenín  3
Affiliations

D-serine released by astrocytes in brainstem regulates breathing response to CO2 levels

S Beltrán-Castillo et al. Nat Commun. .

Abstract

Central chemoreception is essential for adjusting breathing to physiological demands, and for maintaining CO2 and pH homeostasis in the brain. CO2-induced ATP release from brainstem astrocytes stimulates breathing. NMDA receptor (NMDAR) antagonism reduces the CO2-induced hyperventilation by unknown mechanisms. Here we show that astrocytes in the mouse caudal medullary brainstem can synthesize, store, and release D-serine, an agonist for the glycine-binding site of the NMDAR, in response to elevated CO2 levels. We show that systemic and raphe nucleus D-serine administration to awake, unrestrained mice increases the respiratory frequency. Application of D-serine to brainstem slices also increases respiratory frequency, which was prevented by NMDAR blockade. Inhibition of D-serine synthesis, enzymatic degradation of D-serine, or the sodium fluoroacetate-induced impairment of astrocyte functions decrease the basal respiratory frequency and the CO2-induced respiratory response in vivo and in vitro. Our findings suggest that astrocytic release of D-serine may account for the glutamatergic contribution to central chemoreception.Astrocytes are involved in chemoreception in brainstem areas that regulate breathing rhythm, and astrocytes are known to release D-serine. Here the authors show that astrocyte release of D-serine contributes to CO2 sensing and breathing in brainstem slices, and in vivo in awake unrestrained mice.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Fig. 1
Fig. 1
Medullary brainstem astrocytes release d-serine in response to hypercapnia. a, b (Red) d-serine and c, d (red) d-serine racemase were immunodetected in caudal medullary brainstem astrocytes. Merged microphotograph of d-serine and d-serine racemase in GFAP-positive astrocytes (green; b, d) and Hoechst (blue; b, d). e–g d-serine, l-serine, and glutamate were detected by LC-MS/MS in samples of aCSF taken from caudal medullary brainstem (n = 5) or neocortex (n = 3) astrocyte cultures at different time points. Measurements were performed after 30 min under basal conditions (5% CO2), after 5 and 30 min of hypercapnic acidosis (aCSF equilibrated in 10% instead of 5% CO2), and after 30 min of recovery (culture in a 5% CO2 environment). At the end of the experiments, cultures were exposed to 50 mM KCl under basal conditions (5% CO2) for 15 min. P values obtained with Friedman’s test are indicated for each group; multiple comparisons were performed with Conover’s post hoc test; *, **, and *** indicate P < 0.05, P < 0.01, and P < 0.001, respectively; §, #, and &, indicate P < 0.05, P < 0.01, and P < 0.001, respectively for the KCl-evoked increase compared with the experimental conditions represented by each bar, where bar 1 represents the basal conditions. h Effects of hypercapnia (left) and high potassium (right) on d-serine concentration in the incubation medium of medullary astrocytes. Astrocytes were incubated in basal aCSF (n = 7) or calcium-free aCSF (CaCl2 was replaced with MgCl2 in aCSF containing 1 mM EGTA; n = 7) or aCSF containing 10 μM (n = 7) or 100 μM (n = 6) carbenoxolone or 1 mM probenecid (n = 6) or 2 µM bafilomycin A1 (n = 5) or 50 µM brefeldin A (n = 6). Astrocytes were exposed to pharmacological agents for 75 min under normocapnia (5% CO2 equilibrated in air) in basal potassium (3 mM KCl) followed by hypercapnia (10% CO2 equilibrated in air) in basal potassium (4 mM KCl) or by normocapnia in 50 mM KCl for 30 min. d-serine levels attained during hypercapnia or high potassium are expressed as percentage of respective basal levels for each culture at the different conditions. P obtained with Kruskal–Wallis test is indicated; * and ** indicate P < 0.05 and P < 0.01, respectively (Dunn’s multiple comparison post hoc test) between basal aCSF and conditions joined by horizontal lines at the bottom of the figure
Fig. 2
Fig. 2
d-serine increases fR in medullary brainstem slices. a d-serine (10 µM) increased the fR recorded in slices from the ventral respiratory column (indicated in schematic). b, c Concentration–response curves for changes in frequency b and amplitude of the respiratory rhythm c induced by d-serine. The correlation coefficient (R 2) for the best fit to a sigmoidal curve and the number of slices for each concentration are shown. d The increase in fR induced by 10 µM d-serine (red bar) was abolished by 10 µM dizocilpine (MK-801, cross-hatched bar), whereas treatment with MK-801(purple bar) alone did not affect the basal fR (n = 4, P = 0.0156, Friedman’s test; **P < 0.01, Conover post hoc test). e Superfusion for 50 min of medullary brainstem slices with aCSF containing DAAO (n = 9, blue circles), MET-phen (n = 6, cyan circles) or ET-phen (n = 7, brown circles) decreased the fR with respect to controls (n = 5, open circles; F-ratio = 3.08, P = 0.047 mixed ANOVA); * indicates P < 0.05, Bonferroni post hoc test. Symbols and vertical lines indicate the means and SEM, respectively
Fig. 3
Fig. 3
Focal application reveals d-serine sensitive nuclei. a Schematic of a medullary brainstem slice illustrating the sites of injection of d-serine and the extension of the labeled area when a bolus of aCSF containing 1% neutral red dye was pressure-ejected onto a specific locus. b–e Time course of changes in relative fR (ratio between fR at a given time and basal fR) induced by 10 s, 3 psi injections of 300 μM d-serine (red filled circles) and vehicle (aCSF pH 7.4, open circles) in specific brainstem nuclei. Injections of d-serine into the VRC (b) or the RN (c) increased the fR in neonatal medullary brainstem slices, but not when applied to the NTS (d) or the 5-SP (e). The yellow vertical bar indicates when a 10 s pulse was applied. Horizontal dashed line indicates no change. Symbols and vertical lines, mean and SEM, respectively (n = 7 for VRC, RN and NTS; n = 6 for 5-SP). The mixed ANOVA F-ratio and P are indicated for each nucleus; *P < 0.05, **P < 0.01, ***P < 0.001 Bonferroni post hoc test. 5-SP spinal trigeminal nucleus, NTS nucleus tractus solitarius, RN raphe nucleus, RTN retrotrapezoid nucleus, VRC ventral respiratory column
Fig. 4
Fig. 4
d-serine increases fR in awake unrestrained mice. a Ventilatory recording in an adult mouse spontaneously breathing air before (basal) and 60, and 240 min after an i.p. injection of saline (control) or 250 mg kg−1 d-serine. b fR before (basal) and 60 min after an injection of saline (left, n = 6) or d-serine (right, n = 6) for each mouse, represented by colored lines; ns not significant; **, P < 0.01 (Wilcoxon signed-rank test). c Relative fR (ratio between fR at a given time and basal fR) after a single injection of d-serine (F-ratio = 6.39, P = 0.03, mixed ANOVA); **, P < 0.01 Bonferroni post hoc test. d Ventilatory recordings in awake unrestrained mice breathing air before (basal), after 2 min, and after 12 min of 0.2 µl injection of aCSF containing 30 µM d-serine through a cannula implanted and targeted into the RN, under isofluorane anesthesia 4 days before the experiments. e Diagram of the localization of the injection sites into the RN (red spots) visualized with methylene blue dye injected at the end of the experiments. f, g fR of six adult mice before (basal), and 2 min after injection of saline (f) or 30 µM d-serine (g) into the RN. Colored lines connect the fR values before and 2 min after injections for each mouse. ns not significant; * indicates P < 0.05, Wilcoxon signed-rank test. h Time course of the fR in six adult mice before (basal) and after injection of saline (black open symbols and lines) or 30 or 300 µM d-serine (red filled symbols and lines). Time zero corresponds to the injection time. fR is expressed as percentage of basal values (before injection). Dashed horizontal line indicates no change in fR. The mixed ANOVA F-ratio and P are indicated; *P < 0.05, Bonferroni post hoc test
Fig. 5
Fig. 5
d-serine level reduction or NMDAR blockade decreases the respiratory response to hypercapnia in medullary brainstem slices and adult mice. a Integrated inspiratory burst recorded from the VRC in slices obtained from P4 neonates during isohydric normocapnia (basal, 5% CO2, pH 7.4), hypercapnic acidosis (10% CO2, pH 7.2), and recovery to basal conditions. b, d, f, h Exposure to 0.1 U ml−1 DAAO + 260 U ml−1 catalase (b, n = 7) to degrade d-serine, exposure to 50 µM MET-phen (d, n = 6) or 50 µM ET-phen (f, n = 7) to inhibit d-serine racemase, and exposure to 10 µM MK-801 (h, n = 7) for NMDAR blockade for 45 min each reduced the hypercapnia-induced fR response in medullary brainstem slices. Colored lines connect the fR values at pH 7.4 and 7.2 for each medullary brainstem slice before exposure (basal, first column of graphs), after 45 min of exposure (second column of graphs), and after 30 min of wash out (b, recovery). c, e, g, i Average changes in fR induced by hypercapnia under control conditions (basal, white bars); after 45 min of superfusion with aCSF containing DAAO (c, n = 7, blue bar), MET-phen (e, n = 6, cyan bar), ET-phen (g, n = 7, brown bar), or MK-801 (i, n = 7, purple bar); and after 30 min of recovery (second white bar in c). By contrast, 45 min superfusion with basal aCSF (slice responsiveness control) did not modify the fR (j, n = 6) or average responses (k, n = 6). Average data are presented as the mean + SEM (bars and vertical lines, respectively). Analysis for c: P = 0.0062, Friedman’s test; **, P < 0.01 Conover post hoc test. Analysis for b, d–k: ns not significant; * indicates P < 0.05, Wilcoxon signed-rank test. l Ventilatory response to hypercapnia in adult mice injected with saline (control) or saline with 9 mg kg−1 MET-phen. m Average fR response to hypercapnia expressed as the percentage of the basal fR in mice injected with saline (n = 7, controls) and MET-phen (n = 12) at different time points after injection (P = 0.017, mixed ANOVA; *, P < 0.05 Bonferroni post hoc test)
Fig. 6
Fig. 6
Exogenous d-serine restores hypercapnia-induced respiratory response after depression by metabolic inhibition of astrocytes. Integrated inspiratory burst recorded from VRC in eight brainstem slices obtained from P4 neonates and superfused with glutamine (Gln)-supplemented aCSF for 30 min (a, left) then with Gln-supplemented aCSF containing 5 mM fluoroacetate (FA) for 30 min (b, left) and finally with Gln-supplemented aCSF + 5 mM FA + 50 µM d-serine (c, left) during isohydric normocapnia (upper traces, 5% CO2, pH 7.4) and hypercapnic acidosis (lower traces, 10% CO2, pH 7.2). To the right of recordings, colored lines connect the fR values at pH 7.4 and 7.2 for each medullary brainstem slice (n = 8) after 30 min of isohydric normocapnia (pH 7.4) and hypercapnic acidosis (pH 7.2) for 10 min; ** indicates P < 0.01, Wilcoxon signed-rank test; ns not significant. d Average changes in fR induced by hypercapnic acidosis, expressed as percentage of fR values in isohydric normocapnia after 30 min of superfusion with Gln-supplemented aCSF, Gln-supplemented aCSF + FA, and Gln-supplemented aCSF + FA + d-serine (n = 8, P = 0.018, Friedman’s test); multiple comparisons were performed with Conover’s post hoc test; * indicates P < 0.05. e The hypercapnia-induced increment of d-serine levels in 1.5 ml incubation medium containing three caudal brainstem slices was abolished by fluoroacetate (FA). * indicates P = 0.031 (n = 7, Wilcoxon signed-rank test). Changes during hypercapnic acidosis are expressed as percentage of d-serine levels in isohydric normocapnia
Fig. 7
Fig. 7
d-serine released by astrocytes is a mediator of central chemoreception. a The ATP released by astrocytes in response to hypercapnia acts on the purinergic receptors of RTN neurons. The inset shows purinergic receptors in dendrites and the cell soma. b d-serine and glutamate are released by astrocytes of the RN and the VRC in response to hypercapnia. The inset shows synaptic NMDARs. d-serine and glutamate that can also act on extrasynaptic NMDARs in the respiratory network. Thus, d-serine, as a glutamate co-agonist, can modify either the synaptic efficacy by acting on synaptic NMDARs, or the excitability of neurons by acting on extrasynaptic NMDARs. RN raphe nucleus, RTN retrotrapezoid nucleus, VRC ventral respiratory column
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