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Determinants of functional coupling between astrocytes and respiratory neurons in the pre-Bötzinger complex.

Schnell C, Fresemann J, Hülsmann S - PLoS ONE (2011)

Bottom Line: In astrocytes that exhibited rhythmic potassium fluxes and glutamate transporter currents, we did not find a translation of respiratory neuronal activity into phase-locked astroglial calcium signals.We conclude that astrocytes do not exhibit respiratory-rhythmic calcium fluctuations when they are able to prevent synaptic glutamate accumulation.Calcium signaling is, however, observed when glutamate transport processes in astrocytes are suppressed or neuronal discharge activity is excessive.

View Article: PubMed Central - PubMed

Affiliation: Abt. Neuro- und Sinnesphysiologie, Zentrum Physiologie und Pathophysiologie, Georg-August-Universität, Göttingen, Germany.

ABSTRACT
Respiratory neuronal network activity is thought to require efficient functioning of astrocytes. Here, we analyzed neuron-astrocyte communication in the pre-Bötzinger Complex (preBötC) of rhythmic slice preparations from neonatal mice. In astrocytes that exhibited rhythmic potassium fluxes and glutamate transporter currents, we did not find a translation of respiratory neuronal activity into phase-locked astroglial calcium signals. In up to 20% of astrocytes, 2-photon calcium imaging revealed spontaneous calcium fluctuations, although with no correlation to neuronal activity. Calcium signals could be elicited in preBötC astrocytes by metabotropic glutamate receptor activation or after inhibition of glial glutamate uptake. In the latter case, astrocyte calcium elevation preceded a surge of respiratory neuron discharge activity followed by network failure. We conclude that astrocytes do not exhibit respiratory-rhythmic calcium fluctuations when they are able to prevent synaptic glutamate accumulation. Calcium signaling is, however, observed when glutamate transport processes in astrocytes are suppressed or neuronal discharge activity is excessive.

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Rhythmic inward currents in astrocytes of the pre-Bötzinger Complex (preBötC).(A) To identify astrocytes a CCD-image was taken and the astrocyte, identified by its (green) fluorescence in the center of the image was whole-cell recorded in voltage-clamp mode showing (B) respiratory-rhythmic inward currents that were partly obscured by the noise (Vhold = -70 mV; upper trace). The integrated preBötC-field potential (preBötC ∫), recorded in parallel, is shown in the lower trace. (C) Cycle triggered averaging of inward currents was performed, using preBötC-field potentials as triggers to allow the measurement of the amplitude of the respiratory rhythmic current (Iresp,A). (D–F) Input resistance of the astrocytes remains unchanged during astrocytic inward currents: Panels (D) and (E) show whole-cell recordings taken from a fluorescent preBötC astrocyte. (D) Current traces recorded in response to the voltage step protocol, show in the insert, identified this astrocyte as passive. (E) In the presence of bicuculline (20 µM), large amplitude preBötC field potentials were accompanied by large inward currents (asterisks) in the astrocytes (D). Hyperpolarizing voltage steps (−10 mV) were applied to the astrocyte to measure membrane input resistance (Rin), which did not change in association with inward current transients (F; n = 3).
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pone-0026309-g001: Rhythmic inward currents in astrocytes of the pre-Bötzinger Complex (preBötC).(A) To identify astrocytes a CCD-image was taken and the astrocyte, identified by its (green) fluorescence in the center of the image was whole-cell recorded in voltage-clamp mode showing (B) respiratory-rhythmic inward currents that were partly obscured by the noise (Vhold = -70 mV; upper trace). The integrated preBötC-field potential (preBötC ∫), recorded in parallel, is shown in the lower trace. (C) Cycle triggered averaging of inward currents was performed, using preBötC-field potentials as triggers to allow the measurement of the amplitude of the respiratory rhythmic current (Iresp,A). (D–F) Input resistance of the astrocytes remains unchanged during astrocytic inward currents: Panels (D) and (E) show whole-cell recordings taken from a fluorescent preBötC astrocyte. (D) Current traces recorded in response to the voltage step protocol, show in the insert, identified this astrocyte as passive. (E) In the presence of bicuculline (20 µM), large amplitude preBötC field potentials were accompanied by large inward currents (asterisks) in the astrocytes (D). Hyperpolarizing voltage steps (−10 mV) were applied to the astrocyte to measure membrane input resistance (Rin), which did not change in association with inward current transients (F; n = 3).

Mentions: To test for periodic membrane current transients in astrocytes of the preBötC that coincide with rhythmic neuron discharges, we performed whole-cell voltage-clamp recordings from fluorescently labeled astrocytes in the slice preparation. We recorded from a total of 569 fluorescent astrocytes (Figure 1A). As typical, these astrocytes exhibited predominantly passive currents that were distinguished by a linear current-voltage relationship in whole-cell recordings (Figure 1D). Fifty-nine of these astrocytes (10.4%) also exhibited membrane current fluctuations (Iresp,A) that were in phase with the rhythmic discharges of preBötC neurons. Since Iresp,A current amplitude was imbedded to a large extent in background noise (figure 1B), it was not possible to measure current accurately from the raw data. Thus we used cycle triggered averaging to estimate the amplitude, which in 27 astrocytes was –5.9±0.7 pA (mean ± SEM) at Vhold = −70 mV (figure 1C). Iresp,A was recorded as an inward current at clamping potentials between −90 mV and +20 mV (see figure 2A).


Determinants of functional coupling between astrocytes and respiratory neurons in the pre-Bötzinger complex.

Schnell C, Fresemann J, Hülsmann S - PLoS ONE (2011)

Rhythmic inward currents in astrocytes of the pre-Bötzinger Complex (preBötC).(A) To identify astrocytes a CCD-image was taken and the astrocyte, identified by its (green) fluorescence in the center of the image was whole-cell recorded in voltage-clamp mode showing (B) respiratory-rhythmic inward currents that were partly obscured by the noise (Vhold = -70 mV; upper trace). The integrated preBötC-field potential (preBötC ∫), recorded in parallel, is shown in the lower trace. (C) Cycle triggered averaging of inward currents was performed, using preBötC-field potentials as triggers to allow the measurement of the amplitude of the respiratory rhythmic current (Iresp,A). (D–F) Input resistance of the astrocytes remains unchanged during astrocytic inward currents: Panels (D) and (E) show whole-cell recordings taken from a fluorescent preBötC astrocyte. (D) Current traces recorded in response to the voltage step protocol, show in the insert, identified this astrocyte as passive. (E) In the presence of bicuculline (20 µM), large amplitude preBötC field potentials were accompanied by large inward currents (asterisks) in the astrocytes (D). Hyperpolarizing voltage steps (−10 mV) were applied to the astrocyte to measure membrane input resistance (Rin), which did not change in association with inward current transients (F; n = 3).
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pone-0026309-g001: Rhythmic inward currents in astrocytes of the pre-Bötzinger Complex (preBötC).(A) To identify astrocytes a CCD-image was taken and the astrocyte, identified by its (green) fluorescence in the center of the image was whole-cell recorded in voltage-clamp mode showing (B) respiratory-rhythmic inward currents that were partly obscured by the noise (Vhold = -70 mV; upper trace). The integrated preBötC-field potential (preBötC ∫), recorded in parallel, is shown in the lower trace. (C) Cycle triggered averaging of inward currents was performed, using preBötC-field potentials as triggers to allow the measurement of the amplitude of the respiratory rhythmic current (Iresp,A). (D–F) Input resistance of the astrocytes remains unchanged during astrocytic inward currents: Panels (D) and (E) show whole-cell recordings taken from a fluorescent preBötC astrocyte. (D) Current traces recorded in response to the voltage step protocol, show in the insert, identified this astrocyte as passive. (E) In the presence of bicuculline (20 µM), large amplitude preBötC field potentials were accompanied by large inward currents (asterisks) in the astrocytes (D). Hyperpolarizing voltage steps (−10 mV) were applied to the astrocyte to measure membrane input resistance (Rin), which did not change in association with inward current transients (F; n = 3).
Mentions: To test for periodic membrane current transients in astrocytes of the preBötC that coincide with rhythmic neuron discharges, we performed whole-cell voltage-clamp recordings from fluorescently labeled astrocytes in the slice preparation. We recorded from a total of 569 fluorescent astrocytes (Figure 1A). As typical, these astrocytes exhibited predominantly passive currents that were distinguished by a linear current-voltage relationship in whole-cell recordings (Figure 1D). Fifty-nine of these astrocytes (10.4%) also exhibited membrane current fluctuations (Iresp,A) that were in phase with the rhythmic discharges of preBötC neurons. Since Iresp,A current amplitude was imbedded to a large extent in background noise (figure 1B), it was not possible to measure current accurately from the raw data. Thus we used cycle triggered averaging to estimate the amplitude, which in 27 astrocytes was –5.9±0.7 pA (mean ± SEM) at Vhold = −70 mV (figure 1C). Iresp,A was recorded as an inward current at clamping potentials between −90 mV and +20 mV (see figure 2A).

Bottom Line: In astrocytes that exhibited rhythmic potassium fluxes and glutamate transporter currents, we did not find a translation of respiratory neuronal activity into phase-locked astroglial calcium signals.We conclude that astrocytes do not exhibit respiratory-rhythmic calcium fluctuations when they are able to prevent synaptic glutamate accumulation.Calcium signaling is, however, observed when glutamate transport processes in astrocytes are suppressed or neuronal discharge activity is excessive.

View Article: PubMed Central - PubMed

Affiliation: Abt. Neuro- und Sinnesphysiologie, Zentrum Physiologie und Pathophysiologie, Georg-August-Universität, Göttingen, Germany.

ABSTRACT
Respiratory neuronal network activity is thought to require efficient functioning of astrocytes. Here, we analyzed neuron-astrocyte communication in the pre-Bötzinger Complex (preBötC) of rhythmic slice preparations from neonatal mice. In astrocytes that exhibited rhythmic potassium fluxes and glutamate transporter currents, we did not find a translation of respiratory neuronal activity into phase-locked astroglial calcium signals. In up to 20% of astrocytes, 2-photon calcium imaging revealed spontaneous calcium fluctuations, although with no correlation to neuronal activity. Calcium signals could be elicited in preBötC astrocytes by metabotropic glutamate receptor activation or after inhibition of glial glutamate uptake. In the latter case, astrocyte calcium elevation preceded a surge of respiratory neuron discharge activity followed by network failure. We conclude that astrocytes do not exhibit respiratory-rhythmic calcium fluctuations when they are able to prevent synaptic glutamate accumulation. Calcium signaling is, however, observed when glutamate transport processes in astrocytes are suppressed or neuronal discharge activity is excessive.

Show MeSH
Related in: MedlinePlus