Neurons and astrocytes are in close proximity. (A) Projection image from a stack obtained by two-photon laser-scanning microscopy in a slice of a GFAP-eGFP transgenic animal, showing eGFP-positive astrocytes in the MNTB area. Processes of a single astrocyte (arrow) are in contact with at least two principal neurons (indicated by asterisks). Arrowhead indicates eGFP-positive astrocytic endfeet surrounding a blood vessel. Bar, 20 µm. (B) Immunolabeling of a vibratome section from an eGFP mouse with GFAP antibodies coupled with Alexa 594 (red); overlay shows that most eGFP-positive cells (green) are also labeled for GFAP (yellow), although there is no complete overlap (asterisk indicates a GFAP-positive cell that is negative for eGFP). Usually the eGFP signal is more pronounced in the cell body, whereas GFAP labeling is more prominent in processes. The principal neurons appear as hollow dark circles (white arrowheads). Note that the amount of GFAP is much higher in close vicinity to the meninges (bottom right corner). Bar, 20 µm. (C) Overview of an MNTB principal neuron (PN) surrounded by black processes of astrocytes (arrows). (D) Magnified image of outlined area in C. Astrocytes extend their fine processes between the fingers of the calyx of Held (CoH) and the principal neuron (PN). (E and F) Further examples that show how astrocytic processes interdigitate between the fingers of the calyx (CoH) and principal neuron (PN). Black arrowheads indicate active zones (AZ) between a principal neuron and a CoH. Puncta adherentia (PA; white arrowhead) are shown in E. The astrocytic compartments in close vicinity to the calyx contain mitochondria (mit, in F). Note the close apposition of the labeled glial process (white arrow) to the active zone. Bars: (C) 2 µm; (D–F) 0.2 µm.

Spontaneous calcium signals in MNTB astrocytes. (A) Overview of the MNTB region of a GFAP-mRFP1 transgenic mouse brainstem slice showing mRFP1 fluorescence. Bar, 200 µm. (B) On the left, a cell (marked by a box in A) is displayed in higher magnification. The top image is the overlay of mRFP1 expression and Fluo-4 staining, the bottom image is the Fluo-4 staining alone. Bar, 20 µm. On the right, the Fluo-4 fluorescence change (F/F₀) from that cell is shown.

Gliotransmission evokes nSICs in PPNs. (A and A1) Representative paired recording of astrocyte and PPN. Both types of cells were identified morphologically and based on their current profile. aCSF without Ca²⁺ and Mg²⁺ + TTX (1 µM) was used to promote the appearance of nSICs. Astrocytes were dialyzed with 40 mM BAPTA through the pipette and continuous recording of neuron (black trace) and astrocyte were performed for 45–60 min. No nSICs were observed under these experimental conditions (n = 6). (A2) A second neuron was patched after dialysis of the astrocyte (>45 min). This neuron was 20–50 µm away from the original pair recording. Note that no nSICs were observed. (A3) A third neuron located to the right of the original pair recording (>50 µm) showed a nSIC. (B) Summary of nSICs recorded in 3 different neurons (in the same slice) during and after astrocyte dialysis with BAPTA.

Whole-cell recording from a PPN before and after a calcium wave evoked by electrical stimulation. (A) PPNs were recorded under different experimental conditions. nSICs recorded 100 s before and 100 s after an evoked calcium response were used for analysis. Preincubation with the aCSF (alone, modified, or modified + drugs) was always performed. The calcium response was evoked with current pulses (100 µA) at 10 Hz in 4 s. Square shows an nSIC after stimulation. Arrow shows magnification of the recorded nSIC. (B) Calcium response evoked by electrical stimulation. Left: false-color image of the MNTB region. Numbers 1–4 show some active astrocytes. The position of the stimulation electrode is illustrated. Right: calcium responses (F/F₀) of the four astrocytes indicated on the left. (C) Summary of nSICs before and after stimulation in aCSF (n = 16); aCSF + TTX (1 µM) (n = 16); Mg²⁺-free aCSF; Mg²⁺-free aCSF + TTX (1 µM) (n = 17); Mg²⁺-free aCSF + strychnine (1 µM) + gabazine (10 µM) + TTX (1 µM) (n = 20); Mg²⁺-free aCSF + strychnine (1 µM) + gabazine (10 µM) + CNQX (25 µM) + TTX (1 µM) (n = 24); Mg²⁺-free aCSF + strychnine (1 µM) + gabazine (10 µM) + CNQX (25 µM) + ifenprodil (100 µM) (n = 17) + TTX (1 µM); Mg²⁺-free aCSF + strychnine (1 µM) + gabazine (10 µM) + DAAO+ TTX (1 µM) (n = 15); and Mg²⁺-free aCSF + strychnine (1 µM) + gabazine + TTX (1 µM) in a slice where an astrocyte was dialyzed with BAPTA 20 min before (n = 8). See the text and Table S1 for values.

Identification of astrocytes in the MNTB. (A) Fluorescence image showing a principal neuron and a neighboring astrocyte of a GFAP-eGFP mouse, both cells filled with Alexa 594 via the patch pipettes. The top picture shows the red channel, displaying both cells, as well as the tips of both recording pipettes. The middle picture shows the green channel, displaying eGFP fluorescence only in the astrocyte. The bottom picture shows the overlay of eGFP and Alexa fluorescence only in the astrocyte. Bar, 20 µm (refers to all pictures). (B) Representative current profile. Membrane currents were evoked by 50-ms voltage steps ranging from −160 mV to +40 mV, from a holding potential of −70 mV. Note the typical passive response of astrocytes compared with the sodium currents observed in neurons. (C) Current-voltage (IV) relationship from the neuron and the astrocyte shown in B. IV relationship showed a linear pattern in astrocytes and a delayed rectification in neurons. (D) Astrocytes respond to KA and D-Asp. Voltage-clamp recording of an astrocyte clamped to −70 mV. Bath application of KA (0.5 mM) induced inward currents. The response was blocked when CNQX (50 µM) was preincubated and co-applied with KA (0.5 mM). D-Asp (0.5 mM) elicited an inward current too, this response was reduced when TBOA (100 µM) was preincubated and co-applied with D-Asp (0.5 mM). The application bars on the top correspond to all applications. (E) Summary of the effect of CNQX on KA and TBOA on D-Asp currents. The amplitudes of KA and D-Asp currents were normalized, showing the relative reduction induced by CNQX (n = 5) and TBOA (n = 6). Data are the mean ± SEM. Asterisks represent significant differences (*, P = 0.01; **, P = 0.001).

Tetanic stimulation reveals slow inward currents in the astrocytes (aSICs). (A) Currents recorded from astrocytes in response to midline stimulation with 10 pulses at 1 Hz (top), 20 pulses at 10 Hz (middle), and 20 pulses at 100 Hz (bottom). (B) Currents recorded in response to midline stimulation with 20 pulses at 100 Hz. Overlay of three consecutive records from the same cell. Inhibition of glutamate transporters with TBOA (200 µM) results in an increase of the current as compared with control. Application of TBOA (200 µM) and CNQX (20 µM) results in a reduction of the current to almost control value. (C) Summary of the current amplitudes and the decay times of the current induced by 20 pulses at 100 Hz under control conditions, under TBOA and under TBOA together with CNQX (n = 21). Data are the mean ± SEM. Asterisks represent significant difference (**, P = 0.002).

nSICs evoked in PPNs of the MNTB under aCSF without Mg²⁺ and Ca²⁺. (A) Whole-cell voltage-clamp recording from a PPN showing spontaneous nSICs (n = 97 nSICs from 46 neurons) and an mEPSC (asterisk). (B) Representative nSIC (right) and mEPSC (left) recorded in aCSF with TTX (1 µM), strychnine (1 µM), and gabazine (10 µM). (C) Histogram showing amplitude (left) and decay time constant (tau, right) of the recorded nSICs. (D) Cumulative fraction plots of nSICs for amplitude and decay time constant.

Synchrony of nSICs in PPNs is rarely observed. (A) Neuronal paired recording where synchrony of nSICs is absent (n = 10 out of 12 paired recordings). The paired recording was obtained in aCSF with low Ca²⁺ and Mg²⁺ + TTX (1 µM) + strychnine (1 µM) + gabazine (10 µM). (B) Raster plot of a representative neuronal paired recording showing the decay time constant (tau) and time of occurrence of the events recorded in 300 s. A total of 49 events was recorded (events observed in neuron 1 [Δ; 30 events] or neuron 2 [•; 19 events]).