Determinants of Functional Coupling between Astrocytes and Respiratory Neurons in the Pre-Bötzinger Complex

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

Glycinergic interneurons are functionally integrated into the inspiratory network of mouse medullary slices

Neuronal activity in the respiratory network is functionally dependent on inhibitory synaptic transmission. Using two-photon excitation microscopy, we analyzed the integration of glycinergic neurons in the isolated inspiratory pre-Bötzinger complex-driven network of the rhythmic slice preparation. Inspiratory (96%) and ‘tonic’ expiratory neurons (4%) were identified via an increase or decrease, respectively, of the cytosolic free calcium concentration during the inspiratory-related respiratory burst. Furthermore, in BAC-transgenic mice expressing EGFP under the control of the GlyT2-promoter, 50% of calcium-imaged inspiratory neurons were glycinergic. Inspiratory bursting of glycinergic neurons in the slice was confirmed by whole-cell recording. We also found glycinergic neurons that receive phasic inhibition from other glycinergic neurons. Our calcium imaging data show that glycinergic neurons comprise a large population of inspiratory neurons in the pre-Bötzinger complex-driven network of the rhythmic slice preparation

Inhibition of astrocytic glutamate transport elicits robust calcium signals in astrocytes.

<p>Panels (<b>A</b>–<b>D</b>) show an example of fluorometric calcium imaging during glutamate transport: Panel (<b>A</b>) identifies astrocytes, which were loaded with the calcium indicator Oregon Green BAPTA-1 AM (panel <b>B</b>). (<b>C</b>) A blockade of astrocyte glutamate transporters by TFB–TBOA (1 µM) elicited calcium signals in astrocytes (green traces) that were, as shown in (<b>D</b>), not phase-locked to preBötC neuronal activity (preBötC ∫). In the second example (<b>E–G</b>) the effects of glutamate transport inhibition are investigated after mGluR1-blockade. When the incubation of the mGluR1-antagonist CPCCOEt (200 µM) was started 10 min before the application of TFB-TBOA the astrocytic calcium signals were suppressed. (<b>G</b>) Original OGB-1 AM calcium traces are shown from one respiratory neuron (1) and three astrocytes (green traces). Panel (<b>E</b>) shows location of the corresponding EGFP-labeled astrocytes and panel (<b>F</b>) the distribution of the OGB-1-AM labeling.</p

Lack of respiratory-rhythmic calcium signals in astrocytes of the pre-Bötzinger Complex.

<p>The figure shows an example of 2-photon calcium imaging from identified astrocytes in the pre-Bötzinger Complex in the presence of bicuculline (20 µM) and strychnine (10 µM). EGFP astrocytes (<b>A</b>) were labeled with Oregon Green BAPTA-1 AM (OGB-1 AM, <b>B</b>). (<b>C</b>) Cross correlation (CC) maps of OGB-1 AM fluorescence were calculated for each image series between each pixel and a respiratory neuron (cell 7). In panel (<b>D</b>) the OGB-1 AM fluorescence signals from three astrocytes (1–3) and four respiratory neurons (4–7) are depicted with the integrated network output (preBötC ∫). Astrocytes show spontaneous calcium oscillations that were not phase-locked to the neuronal activity. Additionally, as shown in panel (E) the cycle-averaged data of these recording did not reveal any respiratory-rhythmic calcium signal in the astrocytes.</p

Rhythmic inward currents in astrocytes of the pre-Bötzinger Complex (preBötC).

<p>(<b>A</b>) 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>B</b>) respiratory-rhythmic inward currents that were partly obscured by the noise (V_hold = -70 mV; upper trace). The integrated preBötC-field potential (preBötC ∫), recorded in parallel, is shown in the lower trace. <b>(C)</b> 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 (I_resp,A). (<b>D–F</b>) Input resistance of the astrocytes remains unchanged during astrocytic inward currents: Panels (<b>D</b>) and (<b>E</b>) show whole-cell recordings taken from a fluorescent preBötC astrocyte. (<b>D</b>) Current traces recorded in response to the voltage step protocol, show in the insert, identified this astrocyte as passive. (<b>E</b>) 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 (R_in), which did not change in association with inward current transients (<b>F</b>; n = 3).</p

Analysis of respiratory-rhythmic astrocytic currents (I_resp,A).

<p>In panel (<b>A</b>) the effect of BaCl₂ on I_resp,A is shown. Data, for each cell normalized (I/I_max) to the largest current measured over the range of holding potentials, are given for the different holding potentials from −90 mV to +20 mV. Error bars indicate mean ± SEM. The number of cells is indicated below each set of data points. Panel (<b>B</b>) shows cycle-averaged currents (holding potential −70 mV) that were recorded from the rhythmic astrocyte (<b>C</b>) under control conditions, in the presence of barium (BaCl₂, 100 µM) and after additional inhibition of glutamate uptake by dihydrokainate (DHK, 300 µM). The cycle-averaged traces of the corresponding integrated preBötC field potential are depicted underneath.</p

Calcium signals in preBötC astrocytes evoked by activation of mGluR1-receptors.

<p>(A–C) Images show (<b>A</b>) the distribution of astrocytes identified by 900 nm 2-photon excitation and a CFP emission filter (BP 450–500 nm) and (<b>B</b>) Oregon Green BAPTA-1 AM staining <b>(</b>800 nm excitation and BP 511–551 nm emission filter). (<b>C</b>) Fluorescence traces from astrocyte somata shown in panel (A) in presence of DNQX and TTX. Application of quisqualate (5 µM) evoked a robust calcium elevation in 4 out of 5 astrocytes. (<b>D–F</b>) Astrocytic mGluR1-receptor expression is confirmed by immunohistochemistry. Panel (<b>D</b>) shows the confocal image of the EGFP-expressing astrocytes (green), and (<b>E</b>) the mGluR1-receptor expression. The arrows indicate astrocytes that express mGluR1 receptors (Cy-3, red). Note that neighboring neurons also show a high level of mGluR1-expression. In panel (<b>F</b>) the overlay of (<b>D</b>) and (<b>E</b>) is depicted.</p

Astrocytes do not exhibit rhythmic calcium signals.

<p>(<b>A</b>) Current steps evoked in a EGFP-expressing astrocyte by depolarizing and hyperpolarizing voltage steps (10 mV increments) from a holding potential of −70 mV to potentials between −150 to +30 mV. This type of current responses to voltage steps is typical for a passive astrocyte. Panel (<b>B</b>) shows calcium signals (ΔF/F₀) and membrane current (pA) recorded from the particular astrocyte characterized in panel (A), along with simultaneously recorded field potentials (preBötC ∫). In this example, the fluorometric calcium signals (<b>B, Cc</b>) were obtained with Calcium orange (200 µM) loaded via the recording pipette. Rhythmic current fluctuations are buried in the noise but are unmasked by cycle triggered averaging in (C). No phase-locked astrocytic calcium signal could be detected.</p

Glycinergic interneurons in the respiratory network of the rhythmic slice preparation

The neuronal network in the pre-Bötzinger Complex is the key element of respiratory rhythm generation. Isolated in a slice preparation, the pre-Bötzinger Complex network is still able to generate its inspiratory activity. Although the mechanism of rhythm generation in principle relies on glutamatergic neurons, interestingly we found that glycinergic neurons represent a major portion of all inspiratory neurons in the slice preparation