Astrocytic energy metabolism consolidates memory in young chicks.

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 weakly-reinforced 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.

Progressive dysgraphia in a case of posterior cortical atrophy.

Dysgraphia (agraphia) is a common feature of posterior cortical atrophy (PCA). However, detailed analyses of these spelling and writing impairments are infrequently conducted. LM is a 59-year-old woman with dysgraphia associated with PCA. She presented with a two-year history of decline in her writing and dressmaking skills. A 3D T1-weighted MRI scan confirmed selective bi-parietal atrophy, with relative sparing of the hippocampi and other cortical regions. Analyses of LM's preserved and impaired spelling abilities indicated mild physical letter distortions and a significant spelling deficit characterised by letter substitutions, insertions, omissions, and transpositions that was systematically sensitive to word length while insensitive to real word versus nonword category, word frequency, regularity, imagery, grammatical class and ambiguity. Our findings suggest a primary graphemic buffer disorder underlies LM's spelling errors, possibly originating from disruption to the operation of a fronto-parietal network implicated in verbal working memory.

Chicks injected with antisera to either S-100 alpha or S-100 beta protein develop amnesia for a passive avoidance task.

The cellular expression of S-100 beta protein is upregulated in Alzheimer's disease and in Down's syndrome, and this protein has been implicated in memory-related processes in laboratory animals. However, the possibility that the alpha subunit of S-100 is also involved in memory has not yet been examined. In the present study, day-old black Australorp white Leghorn cockerel chicks (Gallus domesticus) received injections of monoclonal antisera to S-100 alpha (1:50) or S-100 beta (1:500) into each hemisphere immediately after training on a one-trial passive avoidance task. The chicks displayed significantly lower retention levels than control birds that had been injected with antisera to carbonic anhydrase, or with saline (p < .01). S-100 alpha antisera had an amnestic effect when injected between 0 and 20 min after training, with memory deficits occurring from 30 min post-learning, at the point of transition between the A and the B phases of the Gibbs-Ng intermediate memory stage. By contrast, the S-100 beta antisera needed to be injected either 5 min before or immediately after training and produced amnesia 10 min earlier, at the start of the A phase of the intermediate memory stage. We conclude that the two subunits of the S-100 protein are required at different points in the sequence of events leading to the consolidation of passive avoidance memory.

Distribution of glutamine synthetase in the chick forebrain: implications for passive avoidance memory formation.

The glial enzyme glutamine synthetase (GS) converts glutamate to glutamine; the latter is used by neurons for the resynthesis of glutamate and GABA. We have used a monoclonal antibody to GS to examine the regional distribution of this enzyme in the forebrains of day-old chicks. GS was detected in glia throughout the rostral and caudal regions of the forebrain and was particularly intense in the hippocampus, area parahippocampus and parts of the hyperstriatal and paleostriatal complex, regions widely considered to be involved in memory formation. Thus, our data provide an anatomical framework for the conclusion that neurons require the support of glia in order to restock their glutamate and/or GABA transmitter supplies during memory processing.

Complex roles of glutamate in the Gibbs-Ng model of one-trial aversive learning in the new-born chick.

Glutamate is the most widespread excitatory transmitter in the CNS and is probably involved in LTP, a neural phenomenon which may be associated with learning and memory formation. Intracerebral injection of large amounts of glutamate between 5 min and 2.5 min after passive avoidance learning in young chicks inhibits short-term memory, which occurs between 0 and 10 min post-learning in a three-stage model of memory formation first established by Gibbs and Ng(25) [Physiol. Behav. 23:369-375; 1979]. This effect may be attributed to non-specific excitation. Blockade of glutamate uptake by L-aspartic and beta-hydroxamate also abolishes this stage of memory, provided the drug is administered within 2.5 min of learning. Interference with either production of percursors for transmitter glutamate in astrocytes or with glutamate receptors is also detrimental to memory formation, but the effects appear much later. After its release from glutamatergic neurons, glutamate is, to a large extent, accumulated into astrocytes where it is converted to glutamine, which can be returned to glutamatergic neurons and reutilized for synthesis of transmitter glutamate, and partly oxidized as a metabolic substrate. The latter process leads to a net loss of transmitter glutamate which can be compensated for by de novo synthesis of a glutamate precursor alpha-ketoglutarate (alpha KG) in astrocytes, a process which is inhibited by the astrocyte-specific toxin fluoroacetate (R. A. Swanson, personal communication). Intracerebral injection of this toxin abolishes memory during an intermediate stage of memory processing occurring between 20 and 30 min post-training (50) [Cog. Brain Res, 2:93-102; 1994]. Injection of methionine sulfoximine (MSO), a specific inhibitor of glutamine synthetase, which interferes with the re-supply of transmitter glutamate to neurons by inhibition of glutamine synthesis in astrocytes, has a similar effect. This effect of MSO is prevented by intracerebral injection of glutamate, glutamine, or a combination and alpha KG and alanine. MSO must be administered before learning, but does not interfere with acquisition since short-term memory remains intact. Administration of either the NMDA antagonist AP5, the AMPA antagonist DNQX, or the metabotropic receptor antagonist MCPF, also induces amnesia. Memory loss in each case does not occur until after 70 min post-training, during a protein synthesis-dependent long-term memory stage which begins at 60 min following learning. However, to be effective, AP5 must be administered within 60 s following learning, MCPG before 15 min post-learning, and DNQX between 15 and 25 min after learning. Together, these findings suggest that learning results in an immediate release of glutamate, followed by a secondary release of this transmitter at later stages of processing of the memory trace, and that one or both of these increases in extracellular glutamate concentration are essential for the consolidation of long-term memory. Since both fluoroacetate and MSO act exclusively on glial cells, the findings also show that neuronal-glial interactions are necessary during the establishment of memory.

Ependymocytes and supra-ependymal axons in rat brain contain glutamate.

The cilated ependymocytes that line the ventricles are decorated by a network of serotoninergic supra-ependymal axons, which are thought to regulate their function. The neurones of origin contain both serotonin and phosphate-activated glutaminase, which raises the possibility that the supra-ependymal axons are also glutamatergic. Using immunocytochemistry, the present study has demonstrated the presence of glutamate in many supra-ependymal axons, as well as in the cilia of ependymocytes. We suggest that glutamate in supra-ependymal axons, counterbalances or opposes the action elicited by serotonin. Glutamate taken up by ependymocytes may supplement metabolic pathways in these cells and could be used to fuel the high energy demands of their cilia.

Inhibition of glutamine synthetase activity prevents memory consolidation.

Methionine sulfoximine, a specific inhibitor of the exclusively glial enzyme glutamine synthetase, was shown, at a concentration of 3.5-4.5 mM, to prevent consolidation of memory for a passive avoidance task in day-old chicks. Provided the drug was administered 5-20 min before the learning task, significant retention loss was observed from the normal time of onset of the second of three postulated stages in the memory formation sequence but the drug had to be administered considerably earlier. The amnestic effect of methionine sulfoximine was successfully counteracted by L-glutamine (10 mM) and monosodium glutamate (4 mM), and also by a cocktail of alpha-ketoglutarate (5 mM) and alanine (5 mM). This effect of methionine sulfoximine is attributed to its blockade of the production of glutamine via the glutamate-glutamine cycle, leading to a reduced capacity of neurons to replenish their transmitter glutamate.

Astrocyte-neuron interaction during one-trial aversive learning in the neonate chick.

During two specific stages of the Gibbs-Ng model of one-trial aversive learning in the neonate chick, we have recently found unequivocal evidence for a crucial involvement of astrocytes. This evidence is metabolic (utilization of the astrocyte-specific energy store, glycogen, during normal learning and inhibition of memory formation by the astrocyte specific metabolic inhibitors, fluoroacetate and methionine sulfoximine) as well as physiological (abolition of memory formation in the presence of ethacrynic acid, an astrocyte-specific inhibitor of cellular reaccumulation of potassium ions). These findings are discussed in the present review in the framework of a more comprehensive description of metabolic and physiological neuronal-astrocytic interactions across an interstitial (extracellular) space bounded by minute processes from either cell type.

Glycogenolytic response of primary chick and mouse cultures of astrocytes to noradrenaline across development.

Glycogen is the brain's largest energy store and it is mainly localised in astrocytes. Glycogen turnover is extremely rapid in the brain, especially during sudden increased demand when glucose supplies are insufficient. Previous culture studies have reported on the glycogenolytic effect of noradrenaline on 3--4 week-old primary mouse astrocyte cultures. This effect is believed to be mediated by the beta-adrenergic-cAMP signal transduction system. Recent evidence has shown a drop in forebrain glycogen levels at a specific time point during memory formation for a passive avoidance task in the day-old chick. This 'memory-related' glycogenolysis may be initiated by noradrenaline-induced rises in cAMP occurring around this point, but it is unknown whether astrocytic glycogenolysis is is stimulated by noradrenaline in day-old chicks. This question was approached in the present study and it was shown that noradrenaline is capable of stimulating both cAMP formation and glycogen breakdown in chick primary astrocyte cultures at developmental age (10-14 days in culture) comparable to the newborn chick. In contrast, noradrenaline did not have a corresponding glycogenolytic effect on 10-day-old mouse astrocyte cultures (equivalent to the 1-week mouse), although it induced a considerable amount of glycogen breakdown in older cultures (18 and 24-26 days).

Astrocytes implicated in the energizing of intermediate memory processes in neonate chicks.

Day-old chicks trained in a single trial passive avoidance task develop three sequentially dependent stages of discrimination memory. The second intermediate stage is made up of two phases: the initial A phase being susceptible to inhibition of oxidative metabolism in the tricarboxcylic acid (TCA) system with 2,4-dinitrophenol (DNP), and a second DNP-insensitive B phase. The studies reported in this paper found that doses of the metabolic toxins fluoroacetate (0.2 mM) and fluorocitrate (0.1 mM) previously reported to disrupt the astrocytic TCA cycle only, also disrupt the A (but not the B) phase of intermediate memory, suggesting an interaction between the astrocytic and neuronal oxidative systems may be required to meet the metabolic demands of this earlier phase. The B phase, on the other hand, was not expressed in the presence of the glycolytic inhibitor iodoacetate (1 mM), suggesting that glycolysis (known to be more efficient in astrocytes) and glycogenolysis (which may be exclusive to astrocytes) may support this second phase of intermediate memory. In this regard, the rise in forebrain noradrenaline levels previously reported to occur before the appearance of the B phase is particularly relevant. Given that noradrenaline has been shown to be capable of enhancing glycogenolysis in astrocyte-enriched cell cultures, it is conceivable that noradrenaline exerts an effect on memory by stimulating the glycolytic system in astrocytes, thereby providing energy or metabolites (e.g. pyruvate) needed to sustain the cellular processes operating during the B phase of intermediate memory.

Astrocytic glycogenolysis energizes memory processes in neonate chicks.

In previous pharmaco-behavioural experiments, we have shown that three sequential stages can be distinguished in discrimination memory for a single trial passive avoidance experience in neonate chicks: a short-term (STM) stage, available for 10 min following learning; an intermediate (ITM) stage, operating between 20 and 50 min (ITMB) post-learning; and a long-term (LTM) stage formed by 60 min after learning. The ITM stage can be divided into two parts: a first phase (ITMA) which is susceptible to inhibition by the uncoupler of oxidative phosphorylation (and thus of oxidative metabolism), 2,4-dinitrophenol (DNP), and a second DNP-insensitive phase (ITMB). ITMA occurs between 20 and 30 min post-training and ITMB between 30 and 50 min. In the present study we have shown: (1) that day-old chicks trained in the passive avoidance task and immediately thereafter injected with the glycolytic inhibitor iodoacetate show retention deficits that are first evident 30 min post-training, and (2) that glycogenolysis, i.e. breakdown of glycogen, a high-molecular carbohydrate energy store localized in astrocytes, occurs in the forebrains of trained, but otherwise untreated birds, between 35 and 55 min after learning. These findings strongly suggest that glycolysis, including astrocytically localized glycogenolysis, is essential to provide energy for active processes occurring during ITMB and that these processes are indispensable for subsequent development of long-term memory.