The astrocytic syncytium represents a multi-cellular network with many cellular and subcellular compartments that can be linked together or separated by regulation of gap junctional connectivity. Because the goal of the present study was to compare overall astrocytic and neuronal lactate trafficking from point sources or from single source cells, assays were carried out in the absence of octanol so that contributions of coupled cells to the response of the reporter cell could be registered as reflecting the total, local astrocytic capacity during brain functional activity. For example, the large volume of the average protoplasmic astrocyte and its extensive arbor of its fine filopodial processes that surround neurons (Bushong et al. 2002) could greatly increase the surface area for glucose delivery from a single astrocyte via many gap junction-coupled astrocytes to a nearby neuron compared to the small cross-section area of the 1 μm diameter micropipette used for focal application of glucose to a neuron (Fig. 6). Note that cell-to-cell transfer distances were measured in our studies but the actual path length traveled by substrates and dyes is not known and may involve various transcellular routes. Another important factor relevant to astrocyte-to-neuron glucose trafficking is the high glucose transport capacity of neurons; it is estimated to be at least nine times that of astrocytes at 5 mmol/L glucose because of kinetic differences between the neuronal glucose transporter (GLUT) 3 (Km = 2.8 mmol/L, kcat = 6512/s) and the astrocytic GLUT1 (Km = 8 mmol/L; kcat = 1166/s) glucose transporters (Simpson et al. 2007). The large surface area of astrocyte-neuron interactions, different Km’s for the respective GLUTs, high glucose level (20 mmol/L) in the micropipette, 30 sec. assay intervals, and avid neuronal uptake of extracellular glucose released from astrocytes may explain the similar glucose uptake rates from a pipette and a more distant astrocyte (Fig. 6). The influence of syncytial trafficking to neuronal glucose uptake and to astrocytic lactate dispersal could be tested in future assays in the presence of octanol.
Astrocytes in the inferior colliculus are highly gap junction coupled, providing extensive intracellular routes that are linked to interstitial fluid via transporters and to perivascular fluid via their endfeet. Syncytial distribution capacity for fuel, metabolites, electrolytes, neurotransmitter precursors, and by-products can be visualized by dye spread (Ball et al. 2007; Rouach et al. 2008) from a single astrocyte to thousands other astrocytes (Fig. 7a) and their perivascular endfeet at some distance from the impaled cell (Fig. 7b). The rapid, high-capacity lactate transport system in astrocytes probably involves diffusion through gap junctions as well as uptake, release, and re-uptake via transporters; in the intact brain this transmembrane substrate ‘cycling’ process would be more extensive than in slices where extracellular material can be washed out into the perfusion solution. In fact, substrate release and washout does affect dye-labeled area in cultured astrocytes; we found that omission of a glucose transporter inhibitor reduced the area labeled by the fluorescent glucose analog 2-NBDG by 56% (see table 1 in Gandhi et al. 2009). A physiological advantage of gap junctional communication plus transmembrane substrate cycling is that extracellular diffusional limitations imposed by dead-space microdomains (Hrabetová and Nicholson 2004) can be circumvented. Figure 7(c) illustrates the concept of preferential astrocytic uptake of lactate generated by glycolytically-activated neurons or astrocytes, lactate cycling across membranes, and distribution of lactate to endfeet where it can be released to perivascular fluid, dispersed along the vasculature, and ultimately released to venous blood. This pathway may enable rapid channeling and preferential release of glucose-derived lactate during brain activation; minimal use of labeled and unlabeled lactate as an oxidative fuel would explain the large underestimates of glucose utilization with [14C]glucose (see introduction). An intriguing implication of astrocyte-mediated trafficking of lactate along the vasculature via perivascular fluid flow (Fig. 7c) is that lactate can modulate blood flow (Laptook et al. 1988; Yamanishi et al. 2006; Gordon et al. 2008), and large syncytia may enable astrocyte-mediated blood flow regulation in a larger tissue volume than that of metabolically-activated cells, thereby helping ensure fuel delivery and by-product removal in excess of demand.
Emerging evidence supports the importance of astrocytic syncytial networks for neuron-astrocyte metabolic interactions (Giaume and McCarthy 1996; Cruz et al. 2007; Ball et al. 2007; Rouach et al. 2008; Tabernero et al. 1996, 2006; Stout et al. 2009; and references cited in these studies), but network metabolite fluxes are not known and some results of syncytial trafficking studies appear to be discordant. For example, we found that astrocytic gap junctional passage of three hexose-6-phosphates is highly restricted compared to anionic fluorescent dyes, hexoses, a triose-phosphate, NADH, and NADPH (Gandhi et al. 2009), whereas Barros et al. (2009) provide evidence for transfer of 2-NBDG-6-P (a fluorescent glucose analog) in cerebellum. The apparent gap junctional permeability of [14C]glucose-6-P in scrape-load assays that measured 14C-labeled area (Tabernero et al. 1996, 2006) probably arose from metabolism of [14C]glucose-6-P by enzymes released from cells damaged by scrape-loading, enabling diffusion of unidentified downstream, gap junction-permeable [14C]metabolites. The importance of astrocyte-to-neuron lactate trafficking in hippocampal slices from 2- to 4-week-old mice was inferred (Rouach et al. 2008) from (i) apparent lactate-dependence of synaptic transmission during assays in the presence of 200 μmol/L 4-CIN and (ii) undetectable astrocyte-to-neuron transfer of 2-NBDG, suggesting that glucose is not passed on to astrocytes and lactate shuttling is necessary to fuel neurons. As noted above, 200 μmol/L 4-CIN completely blocks pyruvate transport and oxidation in isolated mitochondria (Halestrap and Denton 1974, 1975; Halestrap 1975), and in cultured neurons 250 μmol/L 4-CIN reduces lactate and glucose oxidation to 13% and 42% of control rates, respectively (McKenna et al. 2001). Thus, reduced oxidation of glucose-derived pyruvate may have contributed to impairment of synaptic transmission by 4-CIN (Rouach et al. 2008). We observed robust astrocytes-to-neuron glucose shuttling in adult brain slices (Fig. 6) under conditions that favor its detection, i.e., by diffusion of a high concentration of glucose (20 mmol glucose/L) into a single astrocyte; this level would overwhelm its metabolism by endogenous hexokinase and create a strong concentration gradient from the source cell. Further work is necessary to evaluate astrocyte-to-neuron glucose trafficking at lower, normal brain glucose levels and during high neuronal glucose demand, but proof of principle is established. Also, these results suggest that the sensitivity of the 2-NBDG transfer assay, regional differences (i.e., inferior colliculus has higher metabolic rate than hippocampus and would require high, matching transport capacity), and animal age may have contributed to these apparently-discrepant results in 2- to 4-week-old and adult animals. Large, developmental changes in the number, cellular, and regional distributions of brain glucose and lactate transporters, levels of metabolic enzymes, and substrates used for brain fuel (i.e., a shift from utilization of ketones, lactate, and glucose during the suckling period to glucose after weaning (Cremer 1982; Vannucci and Simpson 2003), must be taken into account in studies using cultured cells and tissue from immature animals. Although brain slices have the complex architecture of the brain, they incur preparative damage and lack blood flow. Because arterial pulsations power perivascular fluid flow and tracer distribution in brain (Rennels et al. 1985) metabolite spreading and release from living brain is expected to exceed that in slices. Furthermore, disease states that cause vascular basement membrane thickening (e.g., diabetes and cerebrovascular disease) or blockade of perivascular flow (e.g., Alzheimer’s disease with amyloid plaques in perivascular space) could interfere with fuel distribution within brain and lactate clearance in vivo, thereby influencing brain energetics and interpretation of brain imaging and spectroscopic studies.