Physiological astrocytic calcium levels stimulate glutamate release to modulate adjacent neurons

Abstract

Astrocytes can release glutamate in a calcium-dependent manner and consequently signal to adjacent neurons. Whether this glutamate release pathway is used during physiological signaling or is recruited only under pathophysiological conditions is not well defined. One reason for this lack of understanding is the limited knowledge about the levels of calcium necessary to stimulate glutamate release from astrocytes and about how they compare with the range of physiological calcium levels in these cells. We used flash photolysis to raise internal calcium in astrocytes, while monitoring astrocytic calcium levels and glutamate, which evoked slow inward currents that were recorded electrophysiologically from single neurons grown on microislands of astrocytes. With this approach, we demonstrate that modest changes of astrocytic calcium, from 84 to 140 nM, evoke substantial glutamatergic currents in neighboring neurons (-391 pA), with a Hill coefficient of 2.1 to 2.7. Because the agonists glutamate, norepinephrine, and dopamine all raise calcium in astrocytes to levels exceeding 1.8 microM, these quantitative studies demonstrate that the astrocytic glutamate release pathway is engaged at physiological levels of internal calcium. Consequently, the calcium-dependent release of glutamate from astrocytes functions within an appropriate range of astrocytic calcium levels to be used as a signaling pathway within the functional nervous system.

Figure 1

Single neurons were grown on microislands consisting of purified astrocytes. Neurons were identified by labeling with the fluorescein-conjugated C fragment of tetanus toxin (CFITC; Left), and anti-glial fibrillary acidic protein (GFAP) antibody was used to identify astrocytes (Right).

Figure 2

Photolysis of NP-EGTA increased [Ca²⁺]_i in all astrocytes within single microislands. Cells were coloaded with the calcium indicator fluo-3 and the calcium cage NP-EGTA. (A) The astrocytes of interest were localized by using the fluorescence of fluo-3. (B) After locating the astrocytic microisland of interest, the optical fiber was brought into the field of view. To control the position of UV photolysis, a diode laser, which acted as a searchlight, was enabled. The position of UV pulses corresponds with the position of the red illumination that is shown here. Exposure of the astrocytic microisland to UV pulses delivered through the optical fiber elevated internal calcium level because of photolysis of NP-EGTA. (C) A pseudocolor representation of the resting calcium level. (D) The calcium level after photolysis. Color scale indicates linear pseudocolor representation of fluorescence intensity ranging from 0 to 255 units. (E) The change in [Ca²⁺]_i was also monitored, in a separate experiment, with a photomultiplier tube. Exposure to a train of UV pulses evoked a staircase-like increase in [Ca²⁺]_i, indicating that multiple pulses of UV light can be used to control the level of [Ca²⁺]_i experimentally. Changes in calcium are represented as percentage of ΔF/F_o.

Figure 3

UV photolysis of NP-EGTA evoked an SIC in neurons cultured on astrocytic microislands but not in solitary neurons. Cells were coloaded with fluo-3 and NP-EGTA by bath incubation in the AM esters. To determine the period of dialysis necessary to remove fluo-3 and NP-EGTA from neurons, the fluorescent intensity of fluo-3 was monitored after establishing a whole-cell recording from a solitary neuron (A). In B, the intensity of fluorescence is plotted against time after establishing a whole-cell recording (t = 0). Note that fluorescence signal subsides until it reaches a level corresponding to autofluorescence (dashed line) well within the 10-min period that we adopted as a standard to dialyze NP-EGTA and fluo-3 from neurons in this study. i.u., intensity unit. (C) Neuronal currents recorded after UV illumination in the presence and absence of astrocytes. UV illumination, to photolyse NP-EGTA, caused an SIC in neurons that were cocultured with astrocytes (neuron with astrocytes) but not in solitary neurons. The lightning bolt indicates the onset of the UV stimuli (train of six UV pulses at 20 Hz). Inset summarizes all experiments (n = 8 for neurons with astrocytes; n = 5 for solitary neurons). Bars indicate means ± SEM.

Figure 4

Photolytic elevation of astrocytic calcium causes the NMDA and AMPA receptor-dependent neuronal SIC. Simultaneous recordings of astrocytic calcium levels and neuronal currents indicate that the increase in astrocytic calcium is sufficient to cause glutamate-mediated SIC in neurons. Cells were coloaded with the calcium indicator fluo-3 and the calcium cage NP-EGTA. Both fluo-3 and NP-EGTA were dialyzed out from single neurons on the top of astrocytic islands for 10 min by using a pipette in a whole-cell patch-clamp configuration as indicated by a loss of fluo-3 fluorescence. Astrocytes remained loaded with NP-EGTA that was photolysed by using six UV pulses (20 Hz) delivered through a UV transmitting optical fiber (A, lightning bolt) to cause an increase in astrocytic [Ca²⁺]_i (A, upper trace). Whole-cell recordings from single neurons on top of the astrocytic island indicate that photolysis reliably evoked an SIC (A, lower trace). (B) In contrast, UV stimulation of unloaded astrocytes neither elevated astrocytic calcium nor caused a neuronal SIC. Additionally, D-AP5/CNQX significantly attenuated the ability of photolytic Ca²⁺ elevations in astrocytes to cause neuronal SICs. Changes in astrocytic calcium are represented as percentage of ΔF/F_o. Asterisks indicate significant reduction of measurements as compared with the control group (NP-EGTA +, D-AP5/CNQX −; one-way ANOVA, followed by post hoc Fisher's least significant difference test; P < 0.05).

Figure 5

Simultaneous recordings of astrocytic calcium levels and neuronal currents can be used to elucidate the relationship between astrocytic calcium levels and consequent glutamate release. UV pulses (A, lighting bolts) caused a step-like increase in astrocytic internal calcium (A, upper trace) and an increase in the inward current of adjacent neurons (A, lower trace). (B) The relationships between astrocytic calcium and SIC (and underlying glutamate release from astrocytes) for “all-or-none” (black diamonds) and “graded” (open circles) astrocytes are depicted. These photolysis experiments demonstrate that low levels of calcium are sufficient to induce a neuronal SIC. (C and D) The calcium responses of astrocytes to the application of dopamine (DA) and norepinephrine (NE) (both at 50 μM) demonstrate that the threshold calcium levels necessary to evoke neuronal SICs (dashed lines) are well within the range of the calcium levels that occur in astrocytes. Shaded regions represent astrocytic calcium levels that we have demonstrated are capable of stimulating glutamate release from these nonneuronal cells.