This work was supported by Sniss National Science Foundation Grants 31-39426.93 (A4.T.) and 31-40565.94 (P.J.M.). We thank Drs. S. Poitry, C. Poitry-Yamate, and A.-L. Veuthey, and L. Pellerin and 0. Sorg for their contribution to the experimental work described i n this review: we also thank P. Perrottet and M. Maillard for technical assistance.

The nature of fuel molecules trafficking between mammalian glial cells and neurons was explored using acute retinal cell preparations of solitary Muller glial cells, Muller cells still attached to photoreceptors (the “cell complex”), and solitary photoreceptors. 14C- Molecules in the cell complex, Muller cells, and respective baths were quantitated following 30 min incubation in bicarbonate-buffered Ringer's solution carrying 5 mM 14C(U)-glucose, and substrate preference by solitary photoreceptors was assessed by measuring 14CO2 production. Muller cells alone metabolized 14C-glucose predominantly to carbohydrate intermediates, while the presence of photoreceptors raised proportionately the amount of radiolabeling in amino acids. 14C-Lactate was the major carbohydrate found in the bath. However, in the presence of photoreceptors, its amount was 70% less than that for Muller cells alone. This decrease matched the expected production of 14CO2 by photoreceptor oxidative metabolism and was antagonized by the addition of unlabeled lactate. Moreover, while solitary photoreceptors consumed both exogenous 14C-lactate and 14C-glucose, lactate was a better substrate for their oxidative metabolism. In the cell complex, the metabolism of amino acids increased and illumination affected primarily glutamate and glutamine production: the specific activity of glutamate changed in parallel with that of lactate, and that of glutamine increased by eightfold in darkness. These results demonstrate transfer of lactate from Muller cells to photoreceptors and underscore a photoreceptor-dependent modulation of lactate and amino acid metabolism. We propose that net production and release of lactate by Muller cells serves to maintain their glycolysis elevated and to fuel mitochondrial oxidative metabolism and glutamate resynthesis in photoreceptors.

The retina of honeybee drone is a nervous tissue with a crystal-like structure in which glial cells and photoreceptor neurons constitute two distinct metabolic compartments. The phosphorylation of glucose and its subsequent incorporation into glycogen occur in glia, whereas O2 consumption (QO2) occurs in the photoreceptors. Experimental evidence showed that glia phosphorylate glucose and supply the photoreceptors with metabolic substrates. We aimed to identify these transferred substrates. Using ion-exchange and reversed-phase HPLC and gas chromatography-mass spectrometry, we demonstrated that more than 50% of 14C(U)-glucose entering the glia is transformed to alanine by transamination of pyruvate with glutamate. In the absence of extracellular glucose, glycogen is used to make alanine; thus, its pool size in isolated retinas is maintained stable or even increased. Our model proposes that the formation of alanine occurs in the glia, thereby maintaining the redox potential of this cell and contributing to NH3 homeostasis. Alanine is released into the extracellular space and is then transported into photoreceptors using an Na(+)-dependent transport system. Purified suspensions of photoreceptors have similar alanine aminotransferase activity as glial cells and transform 14C- alanine to glutamate, aspartate, and CO2. Therefore, the alanine entering photoreceptors is transaminated to pyruvate, which in turn enters the Krebs cycle. Proline also supplies the Krebs cycle by making glutamate and, in turn, the intermediate alpha-ketoglutarate. Light stimulation caused a 200% increase of QO2 and a 50% decrease of proline and of glutamate. Also, the production of 14CO2 from 14C-proline was increased. The use of these amino acids would sustain about half of the light-induced delta QO2, the other half being sustained by glycogen via alanine formation. The use of proline meets a necessary anaplerotic function in the Krebs cycle, but implies high NH3 production. The results showed that alanine formation fixes NH3 at a rate exceeding glutamine formation. This is consistent with the rise of a glial pool of alanine upon photostimulation. In conclusion, the results strongly support a nutritive function for glia.

Retinal Müller (glial) cells metabolize glucose to lactate, which is preferentially taken up by photoreceptor neurons as fuel for their oxidative metabolism. We explored whether lactate supply to neurons is a glial function controlled by neuronal signals. For this, we used subcellular fluorescence imaging and either amperometric or optical biosensors to monitor metabolic responses simultaneously from mitochondrial and cytosolic compartments of individual Müller cells from salamander retina. Our results demonstrate that lactate production and release is controlled by the combined action of glutamate and NH(4)(+), both at micromolar concentrations. Transport of glutamate by a high-affinity carrier can produce in Müller cells a rapid rise of glutamate concentration. In our isolated Müller cells, glutamine synthetase (GS) converted transported glutamate to glutamine that was released. This reaction, predominant when enough NH(4)(+) is available, was limited at micromolar concentrations of NH(4)(+), and more glutamate entered then as substrate into the mitochondrial tricarboxylic acid cycle (TCA). Increased production of glutamine by GS leads to increased utilization of ATP, some of which is generated glycolytically. Methionine sulfoximine, a specific inhibitor of GS, suppressed the stimulatory effect of glutamate and NH(4)(+) on glycolysis and induced massive entry of glutamate into the TCA cycle. The rate of glutamine production also determined the amount of pyruvate transaminated by glutamate to alanine. Lactate, alanine, and glutamine can be taken up and metabolized by photoreceptor neurons. We conclude that a major function of Müller glial cells is to nourish retinal neurons and to metabolize the neurotoxic ammonia and glutamate.

SUMMARY1. A double-barrelled potassium-sensitive micro-electrode was developed that was fine enough to record intracellular electrical potentials and potassium activities (aK) in the drone retina.2. aK was measured in the photoreceptor cells, in the pigment (glial) cells, and in the extracellular space, in the superfused, cut, retina. The effect of photostimulation was studied: 20 msec light flashes, intense enough to evoke receptor potentials of maximum amplitude were presented, 1/sec, in a train lasting about 2 min.3. In photoreceptors with membrane potentials 3 50 mV, aK in the dark was 79 mM, S.D. = 27 mM, n = 11. During photostimulation aK fell by 21P5 + 9 5 mMwith a half-time of 30 + 22 sec. (A tentative conversion from activities to free concentrations can be made by taking the activity coefficient as 0 70, its value in the Ringer solution.) 4. In pigment cells with membrane potentials 3 50 mV, aK in the dark was 52 mM, S.D. = 13 mm, n = 11. During photostimulation aK increased by 14 + 5 mM.5. In the extracellular space aK increased during photostimulation with a mean half-time of less than 1-3 sec to a maximum (mean value 14 mM, S.D. = 8-4 mM, n = 22), and then fell to a plateau. 6. It is estimated from the anatomy that the photoreceptors occupy approximately 38 % of the total volume of the retina, the pigment cells 57 %, and the extracellular space 5 %. Hence, it seems possible that during photostimulation nearly all the net loss of potassium from the photoreceptors is temporarily stored in the pigment cells. 7. Recordings were made in the extracellular space of the intact animal by passing the electrode through a hole in the cornea. The mean aK in the dark was 7-7 mM, S.E. = 0.4 mm, n = 22. In the superfused retina, aK in the dark was 6-3 mm, S.E. = 0-7 mm, n = 22, even though aK in the Ringer solution was 2-2 mm. Increasing the aK of the Ringer solution to 7'0 mm had no apparent effect on aK in the extracellular space at depths greater than 20 ,tm.8. In the intact animal the amplitude and time course of the change in extracellular aK evoked by the standard pattern of photostimulation were within the range observed in the superfused preparation.