Predominant enhancement of glucose uptake in astrocytes versus neurons during activation of the somatosensory cortex

Abstract

Glucose is the primary energetic substrate of the brain, and measurements of its metabolism are the basis of major functional cerebral imaging methods. Contrary to the general view that neurons are fueled solely by glucose in proportion to their energetic needs, recent in vitro and ex vivo analyses suggest that glucose preferentially feeds astrocytes. However, the cellular fate of glucose in the intact brain has not yet been directly observed. We have used a real-time method for measuring glucose uptake in astrocytes and neurons in vivo in male rats by imaging the trafficking of the nonmetabolizable glucose analog 6-deoxy-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-aminoglucose (6-NBDG) using two-photon microscopy. During resting conditions we found that astrocytes and neurons both take up 6-NBDG at the same rate in the barrel cortex of the rat. However, during intense neuronal activity triggered by whisker stimulation, astrocytes rapidly accelerated their uptake, whereas neuronal uptake remained almost unchanged. After the stimulation period, astrocytes returned to their preactivation rates of uptake paralleling the neuronal rate of uptake. These observations suggest that glucose is taken up primarily by astrocytes, supporting the view that functional imaging experiments based on glucose analogs extraction may predominantly reflect the metabolic activity of the astrocytic network.

Figure 1.

6-NBDG two-photon microscopy experimental set-up. a, Detail of the cranial window preparation mounted over the barrel cortex. A tungsten wire (50 μm diameter) was inserted close to the imaging area to record the LFP in layer II–III of the barrel cortex. Agarose filled the chamber to stabilize the brain from cardiorespiratory movements. Astrocytes were labeled using SR-101. b, Characterization of 6-NBDG two-photon excitation in vivo. c, Experimental arrangement for simultaneous LFP recording and two-photon imaging during whisker stimulation. 6-NBDG was infused to the femoral vein. The whiskers of the contralateral side were mechanically stimulated by air puffs generating sustained network activity in the LFP. The time-frequency spectrogram of the LFP showed an increase in the LFP power in the 7–20 Hz range during each period of 30 s of whisker stimulation.

Figure 2.

Uptake of 6-NBDG in resting condition. a, Time-lapse sequence of 6-NBDG invading the brain parenchyma (see also supplemental video available at www.jneurosci.org as supplemental material). Green channel shows the blood vessel shape is revealed by 6-NBDG fluorescence at the time of injection. Red channel shows contrast with SR-101 astrocytic staining; neuronal somata appear as dark gaps. Scale bar, 20 μm. b, Kinetic of 6-NBDG uptake in the vascular compartment (B), in an astrocyte (A), and in a neuron (N). Scale bar, 10 μm. c, Astrocytes (in orange) and neurons (in blue) (31 and 30 cells from six animals, respectively) showed similar kinetics of uptake over 45 min.

Figure 3.

Uptake of 6-NBDG during whisker stimulation (WS). a, Fluorescence intensity changes of 6-NBDG in astrocytes, neurons, and blood (red trace, bottom) during functional activation of the barrel cortex. b, Preferential accumulation of 6-NBDG in astrocytes after functional activation. Top, SR-101 astrocytic staining (A for astrocyte and N for neurons); neuronal somata appear as dark gaps. Middle and bottom, Average fluorescence of 6-NBDG over 75 s before and after the stimulation period. Scale bar, 10 μm. c, Slope measurement of 6-NBDG uptake before, during, and after WS. Before WS, both cell types showed a similar slope of 6-NBDG uptake. During WS, only the astrocytic 6-NBDG uptake slope significantly increased (p < 0.05). After WS, the slope uptake returned to a prestimulation level for both cell types. d, Comparison of fluorescence level in cells (astrocytes in orange and neuron in blue) showed that astrocytes take up significantly more 6-NBDG than do neurons (p < 0.001) during functional activation.