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α-Syntrophin is a component of the dystrophin scaffold-protein complex that serves as an adaptor for recruitment of key proteins to the cytoplasmic side of plasma membranes. α-Syntrophin knockout (KO) causes loss of the polarized localization of aquaporin4 (AQP4) at astrocytic endfeet and interferes with water and K+ homeostasis. During brain activation, release of ions and metabolites from endfeet is anticipated to increase perivascular fluid osmolarity, AQP4-mediated osmotic water flow from endfeet, and metabolite washout from brain. This study tests the hypothesis that reduced levels of endfoot AQP4 increase retention of [14C]metabolites during sensory stimulation. Conscious KO and wild-type mice were pulse-labeled with [6-14C] glucose during unilateral acoustic stimulation or bilateral acoustic plus whisker stimulation, and label retention was assayed by computer-assisted brain imaging or analysis of [14C]metabolites in extracts, respectively. High-resolution autoradiographic assays detected a 17% side-to-side difference (p < 0.05) in inferior colliculus of KO mice, not wild-type mice. However, there were no labeling differences between KO and wild-type mice for five major HPLC fractions from four dissected regions, presumably because of insufficient anatomical resolution. The results suggest a role for AQP4-mediated water flow in support of washout of metabolites, and underscore the need for greater understanding of astrocytic water and metabolite fluxes.
Fluxes of glucose through the energy-producing and biosynthetic pathways in neurons and astrocytes are closely linked to rates of neurotransmission. However, the biochemical and physiological processes that underlie increases in glucose utilization during functional activation are not established in sufficient detail to adequately understand the cellular basis of metabolic activity and roles of metabolite trafficking. Brain activation in normal conscious subjects frequently up-regulates glycolysis compared with oxidative metabolism, thereby increasing lactate production by unidentified cells and lactate release from the stimulated structures (Dienel 2012a, b). Rapid release of substantial quantities of lactate underlies the discordant images of brain activation obtained with 14C-labeled deoxyglucose (DG) and glucose (Dienel 2012b), and is consistent with a small magnitude of cell-to-cell lactate shuttling coupled to its oxidation by nearby activated cells, as predicted by metabolic modeling (Mangia et al. 2011).
The [14C]DG method assays the hexokinase step and measures total glucose utilization (Sokoloff et al. 1977), whereas assays with [6-14C]glucose rely on label trapping in the TCA cycle-derived amino acid pools and reflect mainly glucose oxidation. Many laboratories have shown that stimulus-induced increases in total glucose utilization assayed in normal conscious rats with [14C]DG are much higher to those registered with [1- or 6-14C]glucose (Dienel 2012b). Our previous studies have demonstrated that lactate is quickly labeled by [14C]glucose, rapidly dispersed into the astrocytic syncytium via gap junction-mediated metabolite trafficking, and released to blood within minutes of pulse labeling; inhibition of lactate transporters and blockade of gap junctions increase label retention in the activated tissue (Adachi et al. 1995; Cruz et al. 1999, 2007). In adult rat brain slices, astrocytes have a much higher initial rate of and capacity for lactate uptake from extracellular fluid compared with neurons, and lactate diffusion through gap junctions to other astrocytes greatly exceeds lactate shuttling to nearby neurons (Gandhi et al. 2009). Astrocytes are extensively coupled by gap junctions, and dye transfer from a single impaled astrocyte in the inferior colliculus labels as many as 10 000 cells within 5 min and causes extensive dye labeling of gap junction-coupled perivascular endfeet (Ball et al. 2007). Astrocytic endfeet face the vasculature and pia, and contain monocarboxylic acid (Rafiki et al. 2003; Bergersen 2007) and glucose transporters (Simpson et al. 2007), and channels for water (aquaporin4, AQP4) and K+ (Kir4.1 and BK [large-conductance Ca2+-sensitive channels]) (Price et al. 2002; Amiry-Moghaddam et al. 2004a; Nagelhus et al. 2004) (Fig. 1). Release of 14C-lactate and other metabolites to perivascular space facilitates their washout by discharge to blood (Cruz et al. 1999) and the lymphatic drainage system, along with small molecules and proteins (e.g., albumin and amyloid-β) in interstitial fluid (Bradbury and Cserr 1985; Ball et al. 2010).
Figure 1. Model for role of aquaporin4-mediated osmotic water flow in lactate washout during brain activation. The schematic illustrates some nutrient and water fluxes at the interface between the vasculature and astrocytic endfeet. Glucose diffuses down its concentration gradient from blood into brain via the glucose transporter GLUT1 whereas bidirectional co-transport of lactate plus H+ according to its concentration gradient utilizes a monocarboxylic acid transporter (MCT). GLUT1 and MCT1 also co-transport water, with MCT1 carrying 500 water molecules per lactate/H+ and GLUT1 carrying 40 water molecules per glucose; GLUT1 also has a small, passive osmotic water permeability in the presence or absence of glucose (see text). Release of K+ from endfeet during brain activation through Kir4.1 and BK channels has important roles in regulation of blood flow and potassium siphoning associated with neuronal depolarization. Other vasoactive substances released from astrocytic endfeet in response to calcium signals evoked by neuronal activity and enzymes that may influence H+ production and buffering or lactate fluxes (e.g., carbonic anhydrase) are not shown. Glycolysis, lactate production, lactate dispersal among gap junction-coupled astrocytes and their endfeet, and lactate release to cerebral venous blood and perivascular fluid are up-regulated during brain activation. Lactate released to perivascular fluid can stimulate vasodilation. The water channel aquaporin4 (AQP4) is enriched in astrocytic endfeet, and it mediates passive water flow governed by osmotic gradients. Water generated by oxidative metabolism of glucose is thought to be released from brain AQP4. For simplicity and clarity of the schematic, glucose metabolism is portrayed only in the endfoot but major by-products of glucose metabolism (CO2, H2O, and lactate) can be produced in any brain cells and transferred to endfeet for clearance from brain. CO2 formed by oxidative metabolism of glucose can diffuse across membranes or be converted to H+ + HCO3− through the action of carbonic anhydrase, with subsequent buffering of H+ or transport coupled to exchange reactions, e.g., Na+/H+ and HCO3−/Cl− exchangers (not shown). Washout of lactate during brain activation is posited to be supported by AQP4-mediated osmotic water flow and other water fluxes that contribute to the lymphatic drainage system. Knockout of α-syntrophin depletes AQP4 from endfeet and causes its redistribution to other regions of the plasma membrane. Loss of passive water flow at the endfoot is hypothesized to reduce efflux of lactate and other material from activated brain tissue.
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Perivascular fluid flow is driven by aortic pulsations and it moves along the vasculature to spinal lymph nodes and through the cribriform plate of the nose to cervical lymph nodes (Bradbury and Cserr 1985; Rennels et al. 1985; Johnston et al. 2005; Carare et al. 2008; Ball et al. 2010). The peripheral lymphatic drainage system accounts for a large fraction of cerebrospinal fluid (CSF) absorption (Koh et al. 2005; Nagra et al. 2006), and sealing the cribriform plate reduces the passage of [125 I]albumin injected into the lateral ventricle to cervical lymph by 87% (Bradbury and Westrop 1983) and doubles resting intracranial pressure (Mollanji et al. 2002). The lymphatic drainage system is functionally important because ligation or extirpation of cervical lymph nodes causes edema in the brain and eye, increased intracranial pressure, behavioral abnormalities, severe swelling of astrocytic endfeet, mitochondrial swelling, filling of the space between the basement membranes with fluid, appearance of vacuoles in endothelial cells of brain capillaries, myelin damage, increased protein concentration, and elevated macrophage number (Csanda et al. 1963; Csillik and Foldi 1967; Foldi et al. 1967, 1968a,b; Casley-Smith et al. 1976). Together, the above findings indicate that other fluid efflux pathways (e.g., arachnoid villi) do not adequately compensate for lymphatic blockage. The recent, important finding that clearance of mannitol, dextran, and amyloid-β from brain is markedly reduced in AQP4 knockout (KO) mice suggests that AQP4-mediated water flow supports lymphatic drainage and solute removal (Iliff et al. 2012). Differentiated astrocytes cultured from newborn AQP4-null mice have about a two-fold reduction in water permeability when assayed at 37°C (Solenov et al. 2004), raising the possibility that water flux changes of this magnitude may be sufficient to influence tracer clearance in vivo, assuming that astrocytic plasma membrane water permeability is similar in cultured cells and adult brain.
AQP4 is the major brain water channel that is localized in astrocytes, not endothelium, and its polarized distribution at endfeet requires the presence of α-syntrophin, a component of the dystrophin scaffold-protein complex that serves as an adaptor for recruitment of membrane channels, receptors, kinases, and other proteins to the cytoplasmic side of plasma membranes (Albrecht and Froehner 2002; Amiry-Moghaddam et al. 2004a; Bragg et al. 2006; Haj-Yasein et al. 2011). Deletion of the α-syntrophin gene disrupts targeting of AQP4 to endfeet (Yokota et al. 2000; Neely et al. 2001), and AQP4 redistributes to other regions of the astrocytic plasma membrane. AQP4 mis-localization is associated with endfoot swelling during basal conditions, slower clearance of extracellular K+ after electrical stimulation, and attenuated brain edema after transient ischemia or hypo-osmotic challenge (Amiry-Moghaddam et al. 2003a,b, 2004b). Endfoot swelling suggests that tortuosity of extracellular space limits contributions of compensatory water efflux pathways in α-syntrophin KO mice. Endfoot swelling was ascribed to reduced efflux of water generated by glucose oxidation and ATP synthesis, with 6CO2 and 6H2O formed from each glucose and one H2O per ATP formed from ADP + Pi (i.e., 32–36 ATP/glucose) (Amiry-Moghaddam et al. 2003a). However, this magnitude of metabolic water efflux appears to be overestimated because ATP levels are constant under steady state conditions, and an equivalent quantity of water would be consumed by concomitant ATP hydrolysis. Thus, for a resting brain glucose oxidation rate of 1 μmol/g/min, the calculated oxidative water formation rate is 0.11 μL/g/min [i.e., 6 μmol/g/min × 18 μg/μmol × 1 nL/1 μg (density of water) = 108 nL/min/g], which is 30–50% of the rate of interstitial fluid drainage from rat brain [~0.18–0.3 μL/min/g, (Bradbury and Cserr 1985)]. CSF drains from brain via the arachnoid and lymphatic systems (Koh et al. 2005; Nagra et al. 2006), and CSF formation rate in pentobarbital-anesthetized rats is 3.4 μL/min (Chodobski et al. 1995). If astrocytic endfeet are involved in oxidative water and CSF clearance, perivascular fluid-lymphatic water flow may be reduced by AQP4 mis-localization or elimination.
Release of osmotically active compounds (e.g., K+, lactate, and other substances) from astrocytic endfeet into perivascular space during brain activation is posited to stimulate AQP4-mediated osmotic water flow and contribute to lactate washout, and this water-metabolite flux is hypothesized to be reduced in α-syntrophin KO mice (Fig. 1). As a first step toward analysis of factors that contribute to lactate washout from activated brain in vivo, the objective of this study was to determine whether greater quantities of 14C-metabolites of [6-14C]glucose are retained in brain of α-syntrophin KO mice during sensory stimulation. Our previous studies in conscious rats demonstrated that a unilateral broadband acoustic stimulus increased glucose utilization in the activated inferior colliculus, an auditory processing structure, by about 70% when assayed with the routine [14C]DG method (Cruz et al. 2005). A single tone stimulus activates fewer cells and produces a smaller side-to-side difference, about 47% with [14C]DG that contrasts the 18% difference when assayed with [1- or 6-14C]glucose (Cruz et al. 2007). Thus, if AQP4 mis-localization increases retention of labeled metabolites of [14C]glucose, the side-to-side difference should rise from an anticipated 18% in wild-type mice toward 70% in KO mice.
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The biochemical and physiological changes underlying energetics of the neurovascular unit (comprised of astrocytes, neurons, and endothelial cells) during brain activation are complex and inadequately understood. Functional imaging and metabolic studies using [14C]DG, [14C]glucose, [14C]acetate, and other tracers have been useful tools to identify and quantify pathways activated during sensory stimulation of normal conscious rats, and such studies have shown that astrocytes are not solely glycolytic during sensory stimulation; they also increase the fluxes through the glycogenolytic and oxidative pathways (Dienel et al. 2001, 2002, 2007a, b; Cruz and Dienel 2002; Cruz et al. 2005; Dienel 2012b). Our studies with spreading cortical depression demonstrated up-regulation of glycolysis with lactate efflux to blood, and [14C]lactate accounted for almost all of the label released from brain to blood (Adachi et al. 1995; Cruz et al. 1999). However, lactate release to blood explained only half of the magnitude of the 50% underestimate of metabolic rate when assayed with [6-14C]glucose compared with [14C]DG, suggesting the presence of another major route for metabolite washout from brain. Subsequently, we identified lactate transporters and metabolite diffusion through astrocytic gap junctions as key elements in the rapid dispersal of lactate and labeled metabolites from activated regions via the astrocytic syncytium and perivascular endfeet (Ball et al. 2007; Cruz et al. 2007; Gandhi et al. 2009). Recovery of [14C]lactate and other labeled compounds in the meninges after microinfusion of 14C-labeled glucose or lactate into the inferior colliculus, with 34% of the glucose-derived label recovered in the meninges compared with 60% in the infused inferior colliculus, brought our attention to lactate discharge via perivascular-lymphatic drainage system (Ball et al. 2010). Because [6-14C]glucose registers small increases in oxidative metabolism compared with total glucose utilization during activation and lactate does not appreciably accumulate in activated brain, more lactate must be generated and most of the lactate produced must be released (Dienel 2012a, b). Glucose utilization in the inferior colliculus of the rat rose from 0.7 to 1.2 μmol/g/min during broadband acoustic stimulation (Cruz et al. 2005). About 30% of this increase is estimated to be oxidized (0.5 × 0.3 = 0.15) based on trapping of products of [14C]glucose [see Fig. 4d in (Dienel 2012b)], so glycolysis rises by 0.35 μmol glucose/g/min, equivalent to 0.7 μmol lactate/g/min. During 5 min stimulation, 3.5 μmol lactate/g is formed, and brain lactate level rises by ~1 μmol/g, so the quantity of lactate washed out is 2.5 μmol/g, or 0.5 μmol/g/min over the 5 min assay interval. Continuous lactate release from endfeet and lactate transport along the vasculature by pulsatile flow may serve an important long-range signaling function to increase blood flow and glucose delivery to activated tissue because lactate (Hein et al. 2006; Yamanishi et al. 2006; Gordon et al. 2008), K+, and other substances released from endfeet are vasodilators (Attwell et al. 2010; Dunn and Nelson 2010). Release of K+ from BK/Kir4.1 channels to perivascular space is vasodilatory when [K+]e rises from ~3 to < 20 mM (Filosa et al. 2006; Girouard et al. 2010). If K+ release increases perivascular fluid osmolality, water fluxes would rise and may involve endfoot AQP4 channels. MCT-mediated co-transport of water with lactate/H+ released from endfeet and glucose influx from blood would also modulate an endfoot/perivascular space osmotic gradient (MacAulay and Zeuthen 2010).
In this study, we sought to extend our knowledge of brain activation by asking whether disruption of water flow in astrocytic endfeet because of mis-localization of AQP4 in α-syntrophin knockout mice would impair metabolite washout during sensory stimulation (Fig. 1). When total 14C levels in regions of interest in mice given unilateral acoustic stimulation were assayed by brain imaging that has high anatomical resolution, the 14C level was 17% higher in the activated compared with the contralateral inferior colliculus in knockout, but not wild-type, mice (Table 2). Surprisingly, left-right differences were not detected by image analysis in the auditory structures in the wild-type mice (Fig. 2, Table 2), whereas they were readily detected in rats given a similar stimulation protocol (Cruz et al. 2007), raising the possibility that release of labeled metabolites is much faster in mice than rats. Metabolite analysis of extracts of grossly dissected tissue showed that unlabeled glucose levels and the 14C recovered in the glucose fraction in each brain region were similar in the knockout and wild-type mice, suggesting that [14C]glucose does not explain the higher levels of total 14C in autoradiographs from knockout mice (Fig. 2). The biochemical analyses did not, however, reveal lactate accumulation or higher levels of label in downstream pools, such as glutamate, in any of the four structures in the knockout compared with wild-type mice (Table 3). The reasons for discordant results in the imaging and biochemical analysis remain to be established, but inclusion of adjacent tissue with the dissected regions of interest is likely. Accurate dissections of small brain structures in a cryo-glovebox are technically difficult, and the presence of non-activated tissue would blunt and obscure the relatively small differences identified by autoradiographic image analysis.
The similarity of labeling of the five major metabolite fractions and the overall calculated rate of glucose utilization in four brain regions indicates that there are no large differences in metabolism of glucose and metabolite retention in the knockout and wild-type mice (Table 3). However, in both groups, the calculated rates using [6-14C]glucose are much lower than values anticipated from [14C]DG studies, and are similar to values in resting animals. Unilateral sensory stimulation of conscious mice and rats produces side-to-side differences in the range of 50–70% corresponding to rates of up to 1.2 μmol/g/min (Ginsberg et al. 1987; Melzer and Smith 1998; Cruz et al. 2005, 2007; Esaki et al. 2005; Dienel et al. 2007a). Lower calculated rates obtained with [6-14C]glucose (Table 3) compared with [14C]DG and failure to detect side-to-side differences in wild-type mice by brain imaging are consistent with underestimates because of rapid [14C]metabolite efflux in both groups of mice and to use of plasma integrated specific activity.
The brain:plasma distribution ratios for glucose (Fig. 3) are unlikely to have reached a new steady state during the brief activation, but many values, particularly in medial cortex of both groups and visual cortex of wild-type mice were lower than anticipated, based from our previous studies in resting rat brain at steady state (Dienel et al. 1991, 1997; Holden et al. 1991). Mice may have a lower resting steady-state glucose distribution ratio compared with rats, but the lowest ratios probably arose from a glucose supply-demand mismatch during brain activation. Postmortem metabolism of brain glucose is considered an unlikely cause for the low distribution ratios because conversion of glucose to lactate would have increased both the level of unlabeled lactate and the label recovered in the lactate fractions (11.5–12.5 min) above those in the corresponding glucose fractions (3 min), and this was not the case (Table 3). In fact, the lactate fractions contained only about 20–30% of the amount of 14C in the glucose-containing fractions (Table 3). In spite of the lower brain glucose levels in medial cortex compared with the other three regions, the mean tissue glucose concentration was 1 μmol/g (Table 3), which is 20 times the Km of hexokinase for glucose [0.05 mmol/L, (Wilson 2003)]. Thus, even if metabolic demand exceeds supply, glucose is continuously delivered from arterial plasma, which has an adequate concentration of glucose (Table 1) to sustain increased brain metabolism. In rats, glucose delivery exceeds demand by 60% over a wide range of metabolic rates and plasma and brain glucose levels [see Fig. 3e in (Dienel 2012b)]. Based on Michaelis-Menten kinetics, the rate of glucose phosphorylation by hexokinase will not fall until brain glucose level is below 0.5 μmol/g, so the medial cortex still has a two-fold margin of safety.
In conclusion, the higher side-to-side differences detected by autoradiographic brain imaging (Fig. 2, Table 2) raise the possibility that elimination of AQP4-mediated water flow at the astrocytic endfeet is associated with reduced metabolite washout from activated brain. More work is, however, required to establish a role for endfoot water fluxes in perivascular fluid flow and the lymphatic drainage system. Brain water homeostasis is quite complex and poorly understood, and water transport among different compartments is linked to local osmotic changes and water co-transport with lactate, glucose, glutamate, and electrolytes (MacAulay and Zeuthen 2010, 2012). Previous studies with α-syntrophin knockout mice revealed that loss of the polarized localization of AQP4 at endfeet is associated with endfoot swelling and reduced brain edema after transient ischemia or hypo-osmotic challenge, whereas localization of other proteins was not grossly disrupted, e.g., endfoot potassium channel Kir4.1 was stated to be only modestly decreased, and the levels of monocarboxylic acid transporter 1 (MCT1), glucose transporter 1 (GLUT1), and excitatory amino acid transporter 2 (EAAT2) were unaffected (Neely et al. 2001; Amiry-Moghaddam et al. 2003a, b, 2004a). Although knockout of α-syntrophin slowed K+ clearance from extracellular fluid and the mice were more sensitive to hyperthermia-induced seizures, the gene deletion did not alter the amplitude of stimulus-induced population spikes (Amiry-Moghaddam et al. 2003b) or grossly affect obvious behavior or glucose metabolism (Table 3). Deletion of AQP4 influences stimulus-induced extracellular volume dynamics without altering major aspects of synaptic function and plasticity, although burst stimulus-induced long-term potentiation is altered (Skucas et al. 2011; Haj-Yasein et al. 2012).
Four observations, (i) control of water flux at the blood-brain interface by astrocytic endfeet in glial-targeted AQP4-null mice (Haj-Yasein et al. 2011), (ii) swelling of endfeet in the α-syntrophin knockout mice (Amiry-Moghaddam et al. 2003a), (iii) reduced perivascular tracer clearance in AQP4-null mice (Iliff et al. 2012), and (iv) reduced solute clearance, severe swelling of endfeet, and increased intracranial pressure when cervical lymphatic drainage is blocked (Csanda et al. 1963; Foldi et al. 1967, 1968a, b; Casley-Smith et al. 1976; Bradbury and Westrop 1983; Mollanji et al. 2001) suggest that alternative water flux pathways involving arachnoid villi (Nagra et al. 2006) or interstitial fluid flow through clefts between astrocytic endfeet (Mathiisen et al. 2010) and across the vasculature do not adequately compensate for loss of water channels at endfeet and obstruction of lymphatic drainage. Nevertheless, interpretation of the consequences of altered physiology of the α-syntrophin and AQP4-null mice must be conservative and take into account the possibility of contributions from unidentified adaptive changes. For example, deletion of AQP4 reduced the perivascular localization of α-syntrophin and dystrophin (Eilert-Olsen et al. 2012), and knockdown of AQP4 with small interfering RNA reduced expression of connexin43 (Cx43) protein and gap junctional coupling in cultured mouse (but not rat or human) astrocytes, whereas cytoskeletal and morphological changes occurred in rat astrocytes subjected to AQP4 knockdown (Nicchia et al. 2005). Cx43 is a major gap junction protein in astrocytes, and altered Cx43 protein levels and reduced metabolite trafficking throughout the astrocytic syncytium would also reduce rates of metabolite dispersal and discharge during brain activation. In addition, gene knockout or knockdown can cause widespread changes in the brain transcriptome, as shown for Cx43 knockdown or knockout astrocytes. Cx43 down-regulation alters the expression of more than 250 gene products, disrupting intercellular communication and altering the expression (up or down) of genes in many broad categories of cellular function, including transcription, energy and metabolism, cell junctions, adhesion, and extracellular matrix, cell signaling, transport processes, and cell cycle (Iacobas et al. 2005, 2008). Thus, adaptive changes to α-syntrophin or AQP4 gene deletion may contribute to maintaining washout of metabolites from activated brain tissue. Because, the effect of α-syntrophin knockout on retention of labeled metabolites of [6-14C]glucose is measurable but small, new in vivo experimental approaches with high temporal-spatial resolution are needed to assay water and metabolite movement through astrocytes, perivascular fluid, and lymphatic drainage. These methods would also be important to evaluate pathophysiological states, such as diabetes and cardiovascular disease, where structural changes involving the neurovascular unit and thickening of the basement membrane may affect fluid flow and metabolite trafficking in the perivascular-lymphatic system.