Astrocytic energy metabolism consolidates memory in young chicks

Neuroscience 141 (2006) 9 –13

ASTROCYTIC ENERGY METABOLISM CONSOLIDATES MEMORY IN YOUNG CHICKS M. E. GIBBS,a* B. S. O’DOWD,b E. HERTZb AND L. HERTZc

Abstract—In a single trial discrimination avoidance learning task, chicks learn to distinguish between beads of two colors, which are dipped in either a strong or weak tasting aversant (methyl anthranilate) to induce strongly-reinforced and weaklyreinforced learning, respectively. Consolidation of strongly-reinforced learning can be prevented by inhibitors of glycolysis, such as 2-deoxyglucose and iodoacetate and by inhibitors of oxidative metabolism and the consolidation of weakly-reinforced learning can be promoted by administration of glucose. In the present study we show that bilateral, intracerebral injection of 30 nmol acetate can act like glucose to consolidate labile memory and to restore memory impaired by 2-deoxyglucose administration. Acetate is a metabolic substrate that feeds into the tricarboxylic acid cycle, it is oxidized in astrocytes, but not in neurones. Our data suggest that effects of glucose administered 15–25 min post-training on memory consolidation are mediated via astrocytes not neurons. © 2006 IBRO. Published by Elsevier Ltd. All rights reserved.

be excluded in animal experiments where glucose administered by intracerebral microinjection enhances memory (Ragozzino and Gold, 1995). This applies also to bead discrimination learning in day-old chicks, where weakly reinforced learning that normally fades away after 30 min can be consolidated by intracerebral injection of glucose (Gibbs and Summers, 2002a). We have found that acetate, which is metabolized in astrocytes but not in neurones, is also able to promote the memory consolidation occurring 30 min post-training, suggesting that the glucose effect is mediated by astrocytes rather than neurons. In the present experiments we have investigated whether consolidation of memory for bead discrimination in day-old chicks can also be achieved by acetate injection into the intermediate medial mesopallium (IMM), the bird equivalent of the mammalian brain cortex. Acetate is a metabolic substrate that is accumulated and metabolized selectively in the tricarboxylic acid (TCA) cycle in astrocytes (after conversion to acetyl coenzyme A (Acetyl CoA)), whereas it is not oxidatively degraded by neurons (Muir et al., 1986; Waniewski and Martin, 1998). To show that acetate metabolism occurs in astrocytes but not in neurones in day-old chicks we also investigated accumulation of [14C]acetate in primary cultures of chick forebrain neurones and astrocytes at approximately the same developmental stages as in the day-old bird.

Key words: astrocytes, acetate, 2-deoxyglucose, memory consolidation, discriminated bead avoidance, chicks.

EXPERIMENTAL PROCEDURES

a Department of Anatomy and Cell Biology, Monash University, Clayton, Victoria 3800, Australia b Department of Pharmacology, University of Saskatchewan, SK, Canada c

Department of Clinical Pharmacology, China Medical University, Shenyang 110001, PR China

Newly hatched Rhode Island Red⫻New Hampshire male chicks were delivered from a local poultry farm (Wagner’s Poultry, Coldstream, Victoria, Australia) for behavioral experiments. Cell culture experiments were done using fertilized eggs of White Leghorn chicks. Non-radioactive chemicals were from Sigma-Aldrich (St. Louis, MO, USA), and [2-14C]acetate from New England Nuclear (NEN, Boston, MA, USA). All behavioral procedures were approved by the Monash University Animal Ethics Committee and procedures for preparation of cultures by the University of Saskatchewan Animal Ethics Committee and comply with the 1997 Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. All efforts were made to minimize the number of animals used and their suffering. Behavioral experiments used groups composed of eight pairs of chicks (Gibbs and Summers, 2002b). Four 10-s presentations of a small shiny metal bead, followed by a blue, then a red glass bead (4 mm diameter) familiarized the chicks with bead presentations and demonstrated that chicks had no color preference. For the training trial, which commenced 30 min later, the chicks were presented with an identical red bead that had been dipped in either 100% methyl anthranilate to produce strongly-reinforced training (which consolidates to long-term memory where the chicks tend to avoid the red bead) or in 20% anthranilate to produce weakly-reinforced training (which lasts only 30 min before weakening and then the chicks peck both red and blue beads).

Glucose metabolism, measured by positron emission tomography (PET) is reduced in Alzheimer’s disease (Rapoport et al., 1991). In some Alzheimer patients acutely raising glucose levels can facilitate memory (Watson and Craft, 2004). This is consistent with evidence that modest increases in glucose supply can enhance memory (Wenk and Olton, 1989; Korol and Gold, 1998; Messier, 2004). The medium-chain fatty acid octanoate also improves cognitive functions in Alzheimer’s disease (Reger et al., 2004, which is intriguing because octanoate is oxidized in astrocytes (Edmond et al., 1987), although the generated ketone bodies may be metabolized in neurones. Interpretation of memory-enhancing effects of glucose in humans is compounded by the possibility that the effect might be exerted via a hyperglycemia-induced increase in insulin secretion (Hoyer, 2003; Watson and Craft, 2004). This can *Corresponding author. Tel: ⫹61-3-9905-9410; fax: ⫹61-3-9905-8192. E-mail address: [email protected] (M. E. Gibbs). Abbreviations: Acetyl CoA, acetyl coenzyme A; disc.ratio, discrimination ratio; IMM, intermediate medial mesopallium; TCA, tricarboxylic acid; 2-DG, 2-deoxyglucose.

0306-4522/06$30.00⫹0.00 © 2006 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2006.04.038

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M. E. Gibbs et al. / Neuroscience 141 (2006) 9 –13

Memory retention was measured as the ability of the chicks to distinguish between clean red and clean blue beads 120 min post-training. Data were expressed as discrimination ratios (disc. ratio), which is the number of pecks at the blue bead relative to the total pecks at red and blue beads on successive 10 s test trials (Gibbs and Summers, 2002b). When a chick remembers the taste of the previously aversive red bead, it pecks less or avoids the red bead, and the mean disc. ratio approaches 1.0. When a chick does not remember, it pecks equally at red and blue beads and the mean disc.ratio declines toward 0.5. Individual disc.ratios were obtained by computer decoding for each chick and presented as means and S.E.M. for each treatment and test condition.

RESULTS AND DISCUSSION Strongly-reinforced training is consolidated into long-term memory (tested 2 h after training) by metabolic processes occurring 30 min post-training, whereas weakly-reinforced training in young chicks which fades after 30 min is absent at the test after 2 h. Glucose administration can consolidate weakly reinforced learning during the lifetime of the labile memory phase for bead discrimination (before 30 min) (Gibbs and Summers, 2002a). Memory loss following strongly-reinforced training can be produced by central administration of 2-deoxyglucose (2-DG) (Gibbs and Summers, 2002a), which competes with glucose for cellular uptake, but is not glycolytically degraded beyond glucose6-phosphate. Besides inhibiting glycolysis, 2-DG also prevents oxidative metabolism because pyruvate formation is impaired. We have found that administration of lactate can rescue memory consolidation after 2-DG-mediated metabolic inhibition and consolidate weakly reinforced learning (M. E. Gibbs, unpublished observations). Lactate equilibrates with pyruvate, which is oxidatively degraded after introduction into the TCA cycle via acetyl CoA, a process which occurs both in neurones and astrocytes. In the present study we tested whether the astrocyte-specific substrate acetate has similar effects. Sodium acetate (30 nmol) was administered 20 min after training by injection of 5 ␮l of a 6 mM solution in physiological saline (0.9% NaCl) into the left and right IMM, which corresponds to mammalian cortical association areas. Controls were injected with 5 ␮l saline. The location was 2–3 mm left or right of the midline and 3– 4 mm forward of bregma, with the depth of the injection 3.0 – 3.5 mm to the tip of the needle. When 2-DG was applied to inhibit strongly reinforced learning, 250 nmol (25 mM in 10 ␮l volumes), was administered bilaterally into the IMM 5 min before training, whereas controls received physiological saline. If no rescue was attempted, this led to disappearance of memory after 30 min (Gibbs and Summers, 2002a). Cultures of astrocytes were prepared from forebrains of day-14 chicken embryos (O’Dowd et al., 1995) and used when they were 8 –10 days old. These cultures contain no neurones. Cortical neurones were prepared from forebrains of 7-day-old chick embryos as described by Hertz et al. (1989) and used after 2 weeks. These cultures show a minor contamination with astrocytes. For determination of acetate uptake and metabolism the cultures were incubated in serum-free medium containing 50 ␮M sodium

Fig. 1. Amount of acetate accumulated into primary cultures of astrocytes (left) and neurones (right) during 10 min of incubation in serumfree tissue culture medium containing 50 ␮M sodium acetate and trace amounts of [2-14C]acetate and calculated from accumulated radioactivity and protein content per culture (allowing calculation of DPM/mg protein) and the specific activity of the incubation medium (DPM/nmol) by the equation (DPM/mg protein)/(DPM/nmol). Results are means⫾S.E.M. values of six cultures (from two different culture batches) in each group, and acetate uptake in the presence of acetate is statistically significantly different in the two types of cultures (P⬍0.001 in ANOVA, followed by Newman-Keuls test, indicated by an asterisk).

acetate and trace amounts of [2-14C]acetate. Acetate uptake was expressed as nmol/mg protein, calculated as described in legend of Fig. 1. After 10 min of exposure to 50 ␮M acetate more than 5 nmol/mg protein of acetate and its metabolites was present in astrocyte cultures (Fig. 1). Astrocytes in primary cultures have a protein content of ⬃200 nmol protein/g wet wt (Chen et al., 1992). The amount of acetate and its metabolites, mainly the large pools of glutamine and glutamate which are formed from acetate and/or exchange label with ␣-ketoglutarate, an intermediate in the TCA cycle (Sibson et al., 1998; Lebon et al., 2002; Hertz, 2004; Cruz et al., 2005) corresponds to a concentration greater than 1 mmol/g wet wt, or 1 mM. This is a 20 times higher concentration of acetate than in the incubation medium. Since acetate is accumulated by facilitated diffusion up to, but not above the concentration in the medium, such a high concentration can only be achieved by formation of labeled metabolites of acetate, keeping the concentration of nonmetabolized acetate low enough that acetate diffuses into the cells from the medium along a concentration gradient (Hertz and Dienel, 2005). Acetate must therefore have been metabolized at a rate of approximately 0.5 nmol/min per mg protein even at a concentration as low as 50 ␮M. In contrast little acetate uptake was found in neurones (Fig. 1). Diffusional uptake of acetate to a concentration equaling that in the extracellular fluid will, on the same assumptions as above, lead to an uptake of ⬃0.25 nmol/mg protein if no metabolism occurs. Contamination with astrocytes of up to 15% will cause a metabolism-driven uptake of ⬃0.75 nmol/mg protein during the 10-min incubation period. Together these two uptake processes account for

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Fig. 2. Disc. ratio 120 min after training in day-old chickens trained on 20% anthranilate (weakly-reinforced training) with bilateral injection of saline 5 min before training (A) and on 100% anthranilate (strongly-reinforced training) with bilateral injection into IMM of 250 nmol 2-DG 5 min before training (B). Twenty minutes after training the chickens were injected bilaterally in the IMM with either saline or 30 nmol sodium acetate. The saline-injected animals show virtually no learning (disc. ratio close to 0.5), whereas memory was rescued by acetate when the disc. ratio was significantly different, close to 0.9 (P⬍0.001, two-way ANOVA, F1,60⫽45.01). Results are means⫾S.E.M. values for 16 chickens in each group.

all acetate uptake in neuronal cultures, suggesting that there is no acetate metabolism in chicken forebrain neurons, needed to increase the intracellular concentration of labeled compounds to a higher level than in the medium. A possible synthesis of lipids (Kanazawa et al., 1972; Lastennet et al., 1973; Koudinova et al., 2000) must have been quantitatively insignificant and would be energy-requiring rather than energy-producing. Memory after strongly reinforced training was abolished when the animals were treated with 2-DG 5 min before training (Fig. 2B). However, bilateral intracerebral administration of 30 nmol sodium acetate 20 min posttraining rescued memory as indicated by maintained longterm memory 2 h after training (disc. ratio ⬃0.9). This observation suggests that acetate metabolism, which is not affected by 2-DG, is able to substitute for the inhibition of glucose metabolism in the presence of 2-DG, when administered between 15 and 25 min post-training. However, it cannot do so immediately after training, when lactate is capable of rescuing memory (M. E. Gibbs and L. Hertz, unpublished observations). Memory after weakly reinforced training (produced by training with 20% methyl anthranilate) was absent under control conditions (injection of saline at 20 min), as indicated by a disc. ratio close to 0.5 2 h after the training (Fig. 2A). Again, bilateral injection of 30 nmol sodium acetate in the IMM 20 min post-training led to consolidation of longterm memory. However, it is only between 15 and 25 min after training that injection of acetate can achieve consolidation of weakly reinforced learning (Fig. 3). This is a difference from lactate (M. E. Gibbs, unpublished obser-

vations), which is also effective at zero time consistent with previous evidence that glucose metabolism at this time is probably neuronal (Gibbs and Summers, 2002a). It may appear surprising that maintenance of oxidative metabolism in astrocytes can provide memory consolidation after weakly-reinforced training and after inhibition of glucose utilization, but recent observations in intact mammalian brain have indicated that the rate of oxidative metabolism in astrocytes expressed in relation to the volume occupied by these cells is as high as the corresponding rate in neuronal cells (reviewed by Hertz, 2004). Moreover, behavioral training increases the rate of oxidative metabolism in astrocytes in rat brain (Dienel et al., 2003). Also, glucose metabolism in cultured astrocytes, but not in cultured neurones, is increased when the glucose concentration of the incubation medium is raised above its normal level (Walz and Mukerji, 1988; Schousboe et al., 1997), and weakly-reinforced learning can be consolidated by administration of glucose up to 20 min post-training. Since acetate can substitute for glucose during 2-DG inhibition of strongly reinforced learning it was not unexpected that acetate is also able to consolidate weakly-reinforced learning. The character of the missing signal after weakly-reinforced training is unknown, but one possibility could be that it is deficient noradrenaline release and ␤3-adrenergic stimulation of glucose uptake in astrocytes is reduced (Gibbs and Summers, 2002a). It is consistent with this idea that experimental destruction of locus coeruleus, the nucleus of origin for the noradrenergic innervation of the cerebral hemispheres, promotes Alzheimer pathogenesis, reduces glucose utilization and increases memory deficits

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Fig. 3. Consolidation of weakly-reinforced learning promoted by intracortical injection of acetate at specific times after training. F8,131⫽10.39, P⬍0.001 where retention following injection 15–25 min after training was significantly different to saline control, * P⬍0.05. Results are means⫾S.E.M. values for 16 chickens in each group.

in amyloid precursor protein 23 transgenic mice (Heneka et al., 2006). There is also increasing evidence that astrocytic malfunction may play a major role in the development of Alzheimer’s disease (Hertz, 1989; Sastre et al., 2006), and astrocytic cells may become an important target for therapeutic intervention.

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(Accepted 18 April 2006) (Available online 5 June 2006)