Metabolic Interactions between Neurons and Glial Cells

1

Retinal oxygen distribution and consumption

Under normal conditions, O ₂ is the limiting factor in retinal metabolism. Oxygen is used by mitochondria, and their distribution is important in understanding locations of high O ₂ demand. The O ₂ consumption (QO ₂ ) of the retinal pigment epithelial (RPE) cells is about 20% of that of the retina per mg protein. Mitochondria are densely observed in the inner segments (IS) of photoreceptors. Cones have more mitochondria than rods. ^, Mitochondria are also found in each rod spherule and cone pedicle. In the inner retina, the inner plexiform layer has a larger amount of mitochondria than the nuclear layers.

Inner retina

The distribution of oxygen tension (P o ₂ ) close to the vitreo–retinal interface is heterogeneous, being higher close to the arteriolar wall. Preretinal and transretinal P o ₂ profiles indicate that O ₂ diffusion from the arterioles affects the P o ₂ in the juxta-arteriolar areas ( Fig 12.1 ). ^, O ₂ reaches the vitreous by diffusion from the retinal circulation. In contrast, far from the vessels, the preretinal P o ₂ remains constant and the average preretinal P o ₂ from the vitreal side is similar to that measured in the inner retina. In the inner retina, P o ₂ averages about 20 mmHg, but up to 60 mmHg close to the arteriolar wall.

Dark and light O ₂ consumption

Inner retinal oxygen consumption was the same in light and darkness, indicating no influence of light adaptation as there is in the outer retina. In the inner retina, ganglion cells have much higher firing rates if a stimulus is presented repeatedly than if the same amount of light is delivered as a steady background. Consequently, one would expect the inner retina to use more energy when a stimulus is flickering. Indeed, there is, in response to a flickering stimulus, a higher lactate production in inner retina of rabbit than during darkness or steady illumination and a higher deoxy- d -glucose uptake in monkey retina.

Outer retina

Trans-retinal P o ₂ measurements have provided data about the O ₂ supply to the photoreceptors and their QO ₂ . P o ₂ profiles made in cat, ^, pig and monkey indicate that oxygen diffuses from the inner retina and from the choroid towards the middle of the retina, i.e. the outer plexiform layer (OPL). The choroidal circulation supplies about 90% of the photoreceptor's O ₂ use.

Photoreceptor QO ₂ in darkness

In the dark-adapted retina, photoreceptor QO ₂ (Q _OR ) depends strongly on choriocapillary P o ₂ in cat and monkey. ^, In the inner segments of photoreceptors (IS) the local value of QO ₂ is about five times higher than in the outer segments (OS). A similar range for QO ₂ in the outer retina was obtained in rat, rabbit and pig retinas. In both cat and monkey, the average value of P o ₂ in the choriocapillaries is about 50 mmHg, and the corresponding average value of QO ₂ in the outer retina is 4–5 mL O ₂ /100g ⁻¹ /min ⁻¹ . ^,

The average P o ₂ value in cat was 5 mmHg, the minimum was frequently indistinguishable from zero in the dark as predicted by Dollery et al. The amount of oxygen consumed is an indirect measure of ATP synthesis and thus of ATP utilization. The ATP produced in the dark fuels many cellular processes, mainly the Na ⁺ /K ⁺ -ATPase in the IS, which extrudes a large amount of sodium that enters through the light-dependent channels in the OS. An additional process is the turnover of cGMP that holds these channels open. ^,

As noted above, there is no evidence about whether individual rods and cones use different amounts of O ₂ . Cones in the primate fovea appear to use slightly less O ₂ than the parafoveal photoreceptors. ^,

Photoreceptor QO ₂ in light

QO ₂ in the outer retina is lower in steady light than in darkness in various animals investigated. The activity of the Na ⁺ /K ⁺ -ATPase decreases in light, but the turnover of cGMP increases, ^, so the decrease in Q _OR is not as great as the decrease in the pump rate. The maximum size of the overall change appears to be species-dependent.

2

Role of glycolysis underlying retinal function: from whole retina to its parts

Visualization of dynamic functional activity in the retina is an indirect measurement of neuronal activity. It is important to appreciate that any dynamic “functional imaging” of the central nervous system, e.g. fMRI or PET imaging, measures local changes in brain metabolism and physiology that are associated with neuronal activity. Therefore, examining and evaluating retinal function necessarily involves understanding the energy metabolism of this tissue.

The significance of glucose and its metabolism down the glycolytic pathway in mammalian retina is attested to by the measured high rate of aerobic glycolysis in vitro (high capacity for oxygen consumption), ^, its susceptibility to iodoacetate, a strong Pasteur effect (inhibition of glucose utilization) and the aerobic and anaerobic production of lactate.

The adult retina, as is the case for every CNS region, depends on an uninterrupted supply of blood-borne glucose. Under normal conditions, glucose is virtually the sole substrate supporting the intense energy metabolism required to maintain retinal function, e.g. normal electrical responsiveness to light and neurotransmission. Lactate generated from either anaerobic glycolysis or glycogenolysis within the retina has recently been proposed as another important energy source during synaptic transmission, but its uptake and metabolism by retinal cells has yet to be demonstrated in vivo. A critical evaluation of lactate use in the CNS has been presented.

The distribution of key enzymes in glucose metabolism through the individual neuronal and synaptic retinal layers and the dehydrogenases for several of the intermediate stages of glucose degradation in retina have been documented in a series of pioneering histochemical work and set the stage early for later metabolic studies of the glycolytic and oxidative capacity of this tissue. Kuwabara and Cogan's studies suggested for the first time that the Müller glial cells may serve a significant metabolic role, in stark contrast to the general view at that time as having only a structural function. Indeed, of the two major cell types in retina – the Müller glia and photoreceptors – metabolic studies have demonstrated that Müller cells both in situ and when acutely isolated, preferentially and massively take up and phosphorylate glucose, part of which is stored as glycogen. ^, Further evidence to confirm this finding has been performed in vivo.

More recent supporting evidence comes from the use of iodoacetate (IAA), a well-known glycolytic poison that exerts its effect when the transformation of glucose is committed to proceed through glycolysis. In the 1950s, it was shown that intravenous delivery of sodium iodoacetate in rabbit, monkey and cat abolished the electrical response of the visual pathway to illumination within minutes, with a resulting histological picture similar to that presented in human retinitis pigmentosa. This led to the speculation that the initial effect must be on the visual cells, although Warburg suggested that the different cell types in the retina may not contribute equally to the general biochemical picture. Indeed, this suggestion has been unambiguously supported by the differential suppression of the component waves of the extracellular electroretinogram (ERG). ^, However, beyond this evidence, the identity of the retinal cell types taking up IAA remains unknown. Recently this was explored using synchrotron-based x-ray fluorescence of IAA at the cellular level in situ ( Fig. 12.2 ). The fluorescence map ( Fig. 12.A ) generated from the dark-adapted retina ( Fig. 12.2B ) showed that IAA was taken up specifically by Müller glia and not by retinal neurons, including photoreceptors, indicating that the effect of IAA on neurons is not direct but secondary to inhibiting glycolysis in glia (Poitry-Yamate 2009, unpublished results). Together, these results suggest a key role played by Müller glia in transporting glucose from the blood into the retina.

Acutely isolated mammalian photoreceptors produce ¹⁴ CO ₂ from ¹⁴ C(U)-glucose while photoreceptor outer segments produce both lactate from glucose and ¹⁴ CO ₂ from ¹⁴ C(U)-glucose. These authors interpreted their results as indicating that both glycolysis and the pentose phosphate pathway contribute to maintaining photoreceptor function. Considering that only one in six carbons from ¹⁴ C(U)-glucose is converted to ¹⁴ CO ₂ through the pentose phosphate pathway, and the abundance of photoreceptor mitochondria, it is likely that the ¹⁴ CO ₂ reflects the rate of mitochondrial respiration. Photoreceptors of some species do not express Gpi 1, the enzyme catalyzing the isomerization of glucose-6-phosphate to fructose-6-phosphate, leaving the phosphate pentose pathway as the only possible downstream path, with a gain of 2 NADPH molecules potentially serving as reducing agent for the reduction of retinaldehyde.

A number of studies of glucose metabolism have been undertaken in intact retinal tissue or with acutely isolated cell models in vertebrate/mammalian retina. Of these, one is unique for studying not only glucose metabolism but also metabolic compartmentation: the cell model of acutely isolated Müller cells still attached to photoreceptors (termed “the cell complex”) shown in Figure 12.3 and further discussed in sections 4, 6 and 8. This study confirmed not only the previous work by Kuwabara and Cogan, but showed for the first time in a mammalian preparation of the CNS tissue that glial cells transform rather than simply transfer the primary energy substrate glucose and supply neurons with a glucose-derived metabolite.

Cell culture models of transformed rat Müller cells, human RPE and transformed mouse photoreceptor cells and ganglion cells were all found to produce lactate, aerobically and anaerobically in the presence of 5 mM glucose. This may not be surprising as culturing techniques influence cell metabolism and function. The composition of culturing medium may be a key underlying factor to explain why cells are predominantly glycolytic, irrespective of cell type. However, the production of lactate by these cells did not significantly differ with the addition of 10 mM lactate. In general, lactate production and its release into the extracellular space as a lactate anion plus a proton (H ⁺ ) creates an extracellular pH gradient, i.e. increased proton concentration and consequently pH values become less than 7.4. Depending on the magnitude, direction and time course of the extracellular pH gradient, lactate may accumulate extracellularly, or alternatively, lactate may be taken up on a proton-linked monocarboxylate transporter. In this context, transformed, cultured neurons did not transport or metabolize exogenous lactate, consistent with a lactate-containing solution adjusted to a pH of 7.4 rather than a pH of <7.4.

3

Biochemical specialization of glial cells

The major glial cell type in vertebrate retina is the radial Müller glial cell (also termed radial fibers or sustentacular cells of Heinrich Müller). Structurally, they are elongated, possess a prominent specialized region called endfeet at the inner limiting membrane, and are vertically oriented with respect to the retinal layers ( Fig. 12.4 ). Since Müller cells extend through the synaptic and nuclear layers of the retina from the inner to outer limiting membranes they are in intimate apposition to every neuron cell type. Müller glia also serve as an additional physical and functional cell layer to the diffusion of substances into and out of the extracellular space, the vitreous, the subretinal space and retinal vascular supply. Histochemical evidence has shown that glycogen synthesis, glycogenolysis and anaerobic glycolysis are localized to Müller glial cells in situ. This was confirmed and quantitated in living intact, dark-adapted retina using biochemical and autoradiographic methodologies ^, and provided strong experimental evidence for the working hypothesis of all retinal cell types, Müller glia play a major role beyond the blood–retinal barriers in transporting glucose from the blood into the neural retina. However, once in the neural retina, it remains to be shown along the Müller cell's entire radial length whether the distribution of transporters related to energy substrate uptake and release are tailored to this cell's own metabolic needs, but yet adapted to the function and metabolic needs of their immediate neuronal environment.

Two functional and biochemical specializations unique to Müller glia are their capacity to inactivate the excitatory neurotransmitter glutamate ^, and inhibitory neurotransmitters GABA and glycine. They are the exclusive and/or predominant cellular site of:

• (1)

  glutamine synthetase activity for the synthesis and release of glutamine, a precursor for photoreceptor neurotransmitter resynthesis; and

• (2)

  carbonic anhydrase for the conversion of water and CO ₂ of neuron origin to bicarbonate, an enzymatic activity implicated in the regulation of intracellular and extracellular pH and volume.