Role of the Astrocytic Na(+), K(+)-ATPase in K(+) Homeostasis in Brain: K(+) Uptake, Signaling Pathways and Substrate Utilization.

This paper describes the roles of the astrocytic Na(+), K(+)-ATPase for K(+) homeostasis in brain. After neuronal excitation it alone mediates initial cellular re-accumulation of moderately increased extracellular K(+). At higher K(+) concentrations it is assisted by the Na(+), K(+), 2Cl(-) transporter NKCC1, which is Na(+), K(+)-ATPase-dependent, since it is driven by Na(+), K(+)-ATPase-created ion gradients. Besides stimulation by high K(+), NKCC1 is activated by extracellular hypertonicity. Intense excitation is followed by extracellular K(+) undershoot which is decreased by furosemide, an NKCC1 inhibitor. The powerful astrocytic Na(+), K(+)-ATPase accumulates excess extracellular K(+), since it is stimulated by above-normal extracellular K(+) concentrations. Subsequently K(+) is released via Kir4.1 channels (with no concomitant Na(+) transport) for re-uptake by the neuronal Na(+), K(+)-ATPase which is in-sensitive to increased extracellular K(+), but stimulated by intracellular Na(+) increase. Operation of the astrocytic Na(+), K(+)-ATPase depends upon Na(+), K(+)-ATPase/ouabain-mediated signaling and K(+)-stimulated glycogenolysis, needed in these non-excitable cells for passive uptake of extracellular Na(+), co-stimulating the intracellular Na(+)-sensitive site. A gradual, spatially dispersed release of astrocytically accumulated K(+) will therefore not re-activate the astrocytic Na(+), K(+)-ATPase. The extracellular K(+) undershoot is probably due to extracellular hypertonicity, created by a 3:2 ratio between Na(+), K(+)-ATPase-mediated Na(+) efflux and K(+) influx and subsequent NKCC1-mediated volume regulation. The astrocytic Na(+), K(+)-ATPase is also stimulated by ß1-adrenergic signaling, which further stimulates hypertonicity-activation of NKCC1. Brain ischemia leads to massive extracellular K(+) increase and Ca(2+) decrease. A requirement of Na(+), K(+)-ATPase signaling for extracellular Ca(2+) makes K(+) uptake (and brain edema) selectively dependent upon ß1-adrenergic signaling and inhibitable by its antagonists.

Antagonists of the Vasopressin V1 Receptor and of the ß(1)-Adrenoceptor Inhibit Cytotoxic Brain Edema in Stroke by Effects on Astrocytes - but the Mechanisms Differ.

Brain edema is a serious complication in ischemic stroke because even relatively small changes in brain volume can compromise cerebral blood flow or result in compression of vital brain structures on account of the fixed volume of the rigid skull. Literature data indicate that administration of either antagonists of the V1 vasopressin (AVP) receptor or the ß1-adrenergic receptor are able to reduce edema or infarct size when administered after the onset of ischemia, a key advantage for possible clinical use. The present review discusses possible mechanisms, focusing on the role of NKCC1, an astrocytic cotransporter of Na(+), K(+), 2Cl(-) and water and its activation by highly increased extracellular K(+) concentrations in the development of cytotoxic cell swelling. However, it also mentions that due to a 3/2 ratio between Na(+) release and K(+) uptake by the Na(+),K(+)-ATPase driving NKCC1 brain extracellular fluid can become hypertonic, which may facilitate water entry across the blood-brain barrier, essential for development of edema. It shows that brain edema does not develop until during reperfusion, which can be explained by lack of metabolic energy during ischemia. V1 antagonists are likely to protect against cytotoxic edema formation by inhibiting AVP enhancement of NKCC1-mediated uptake of ions and water, whereas ß1-adrenergic antagonists prevent edema formation because ß1-adrenergic stimulation alone is responsible for stimulation of the Na(+),K(+)-ATPase driving NKCC1, first and foremost due to decrease in extracellular Ca(2+) concentration. Inhibition of NKCC1 also has adverse effects, e.g. on memory and the treatment should probably be of shortest possible duration.

Rapid turnover of glycogen in memory formation.

The influence of noradrenaline acting at α(2)-AR and ß(2)-ARs on the turnover of glycogen after learning has been investigated. The role of glycogen turnover in memory formation was examined using weakly-reinforced, single trial bead discrimination training in day-old domestic chickens. This study follows our previous work that focused on the need for glycogen breakdown (glycogenolysis) during learning. Inhibition of glycogenolysis by 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) prevented the consolidation of strongly-reinforced learning and inhibited memory. The action of DAB could be prevented by stimulating glycogenolysis with the selective ß(2)-AR agonist, zinterol. Stimulation of α(2)-ARs has been shown to lead to an increase in the turnover and synthesis of glycogen. In the present study, we examined the effect of inhibition of α(2)-AR stimulated glycogen turnover (measured as(14)C-glucose incorporation into glycogen) on the ability of zinterol to promote the consolidation of weakly reinforced memory. In astrocytes, the selective α(2)-AR agonist clonidine stimulated (14)C-glucose incorporation into glycogen in chick astrocytes and this was inhibited by the selective α(2)-AR antagonist, ARC239. The critical importance of the timing of ARC239 injection relative to training and intracerebral administration of zinterol was examined. It is concluded that our data provides evidence for a readily accessible labile pool of glycogen in brain astrocytes. If glycogen synthesis is inhibited, the can be depleted within 10 min, thus preventing zinterol from promoting consolidation.

α2-Adrenoceptors activate noradrenaline-mediated glycogen turnover in chick astrocytes.

In the brain, glycogen is primarily stored in astrocytes where it is regulated by several hormones/neurotransmitters, including noradrenaline that controls glycogen breakdown (in the short term) and synthesis. Here, we have examined the adrenoceptor (AR) subtype that mediates the glycogenic effect of noradrenaline in chick primary astrocytes by the measurement of glycogen turnover (total (14) C incorporation of glucose into glycogen) following noradrenergic activation. Noradrenaline and insulin increased glycogen turnover in a concentration-dependent manner. The effect of noradrenaline was mimicked by stimulation of α(2) -ARs (and to a lesser degree by ß(3) -ARs), but not by stimulation of α(1) -, ß(1) -, or ß(2) -ARs, and occurred only in astrocytes and not neurons. In chick astrocytes, studies using RT-PCR and radioligand binding showed that α(2A) - and α(2C) -AR mRNA and protein were present. α(2) -AR- or insulin-mediated glycogen turnover was inhibited by phosphatidylinositol-3 kinase inhibitors, and both insulin and clonidine caused phosphorylation of Akt and glycogen synthase kinase-3 in chick astrocytes. α(2) -AR but not insulin-mediated glycogen turnover was inhibited by pertussis toxin pre-treatment indicating involvement of Gi/o proteins. These results show that the increase in glycogen turnover caused by noradrenaline is because of activation of α(2) -ARs that increase glycogen turnover in astrocytes utilizing a Gi/o-PI3K pathway.

Inhibition of Abeta aggregation and neurotoxicity by the 39-kDa receptor-associated protein.

Aggregation of beta-amyloid protein (Abeta) to form oligomers is considered to be a key step in generating neurotoxicity in the Alzheimer's disease brain. Agents that bind to Abeta and inhibit oligomerization have been proposed as Alzheimer's disease therapeutics. In this study, we investigated the binding of fluorescein-labeled Abeta(1-42) (FluoAbeta(1-42)) to SH-SY5Y neuroblastoma cells and examined the effect of the 39-kDa receptor-associated protein (RAP), on the Abeta cell interaction. FluoAbeta(1-42) bound to the cells in a punctate pattern. Surprisingly, when RAP was added to the incubations, FluoAbeta(1-42) and RAP were found to be co-localized on the cell surface, suggesting that RAP and Abeta may bind to each other. Experiments using the purified proteins confirmed that a RAP-Abeta complex was stable and resistant to sodium dodecyl sulfate. RAP also inhibited Abeta oligomerization. We next examined whether RAP could inhibit the neurotoxic effects of Abeta. Addition of Abeta(1-42) to SH-SY5Y cells caused an increase in intracellular Ca2+ that was inhibited by treatment of the Abeta peptide with RAP. RAP also blocked an Abeta-induced inhibition of long-term memory consolidation in 1-day-old chicks. This study demonstrates that RAP binds to Abeta and is an inhibitor of the neurotoxic effects of Abeta.

What learning in day-old chickens can teach a neurochemist: focus on astrocyte metabolism.

The learning process sets in motion a prolonged, reproducible, and complicated pattern of brain activation, which provides information about biochemical reactions in activated brain. Study of this pattern during one-trial aversive bead discrimination in day-old chick is facilitated by precise timing of sequential metabolic events occurring between a 10-s learning period, in which the chicks learn to associate a red bead with aversive taste, and memory consolidation, indicated by unwillingness to peck at untainted red beads while freely pecking at corresponding blue beads. Inhibition of learning by metabolic inhibitors and restoration of memory by specific substrates at specific times allow determination of specific metabolic events and their neuronal or astrocytic localization. Downstream metabolism of glycogen and of glucose to pyruvate/lactate is segregated into separate pools. Glucose metabolism via pyruvate dehydrogenation provides energy in both neurons and astrocytes and may include gap junction-mediated lactate transport into astrocytes. A key role is played by glycogenolysis, stimulated by beta2-adrenergic and/or 5-HT2-receptor stimulation along with alpha2-adrenergic stimulation of glycogen synthesis. The importance of glycogen reflects that it selectively supports de novo synthesis of transmitter glutamate by combined pyruvate dehydrogenation and carboxylation in astrocytes.

Rescue of Abeta(1-42)-induced memory impairment in day-old chick by facilitation of astrocytic oxidative metabolism: implications for Alzheimer's disease.

Administration of small oligomeric beta-amyloid (Abeta)(1-42) 45 min before one-trial bead discrimination learning in day-old chicks abolishes consolidation of learning 30 min post-training (Gibbs et al. Neurobiol. Aging, in press). Administration of the beta3-adrenergic agonist CL316243, which specifically stimulates astrocytic but not neuronal glucose uptake, rescues Abeta impaired memory. Weakly reinforced training can be consolidated by various metabolic substrates and we have demonstrated neuronal dependence on oxidative metabolism of glucose soon after training versus astrocytic glucose dependence 20 min later. Based on these findings we examined whether different metabolic substrates were able to counteract memory inhibition by Abeta(1-42). Although lactate, the medium-chain fatty acid octanoate, and the ketone body beta-hydroxybutyrate consolidated weakly reinforced training when injected close to learning, none of them were able to salvage Abeta-impaired memory; at this early time. All three metabolites and the astrocytic-specific acetate consolidated weak learning and rescued Abeta-impaired memory when injected 10-20 min post-training. However, neither glucose nor insulin rescued memory when injected at 20 min. Rescue of memory by providing astrocytes with alternative substrates for oxidative metabolism suggests that Abeta(1-42) exerts its amnestic effects specifically by impairing astrocytic glycolysis.

Astrocytes and interneurons in memory processing in the chick hippocampus: roles for G-coupled protein receptors, GABA(B) and mGluR1.

Glutamate and GABA acting at mGluR1 and GABA(B) receptors, respectively, have roles in memory processing in the hippocampus up to 35 min after bead discrimination learning in the young chick. Activation of mGluR1 receptors is important at 2.5 and 30 min after training, but modulation of these receptors between these two times has no effect on memory. This timing is similar to the action of glutamate on NMDA receptors. The GABA(B) antagonist, phaclofen, and the inhibitor of astrocytic oxidative metabolism, fluoroacetate, inhibited memory when injected between 2.5 and 30 min. Paradoxically, a high dose of the GABA(B) agonist, baclofen, also inhibited memory, but a low dose promoted memory consolidation--an effect possibly caused by too much information and loss of the 'message'. These results are interpreted in terms interactions between interneurons, astrocytes and pyramidal cells and demonstrate the importance of all cell types in memory processing in the hippocampus.

Importance of adrenergic receptors in prenatally induced cognitive impairment in the domestic chick.

In the domestic chick, mild hypoxia (24h of 14% oxygen) at two stages of embryonic development results in post-hatch memory deficiencies tested using a discriminated bead avoidance task. The nature of the memory loss depends on the gestational age at which the hypoxia occurs. Hypoxia on embryonic day 10 (E10) of a 21 day incubation results in chicks with no short-term memory 10 min after training, whereas hypoxia on day 14 (E14) results in chicks with good labile memory 30 min after training but no consolidation of memory into permanent storage (120 min). Hypoxia at E14 is associated with increased plasma levels of noradrenaline and therefore we suggest that altered catecholamine exposure within the brain contributes to cognitive problems by modifying the responsiveness of brain beta-adrenoceptors. In ovo administration of noradrenaline, or the beta(2)-adrenoceptor agonist formoterol, at E14 had the same effect on memory consolidation as hypoxia. Following hypoxia at E14, memory could be rescued after training by central injection of a beta(3)-adrenoceptor agonist, but not by a beta(2)-adrenoceptor agonist. The differences in the responsiveness of memory processing to beta(2)-adrenoceptor agonists suggests alterations to the receptors or downstream of the receptor activation. However, both types of beta-adrenoceptor agonists rescued memory in E10 treated chicks implying that at this age hypoxia does not affect the receptors. In summary, hypoxia or increased levels of stress hormones during incubation alters beta-adrenoceptor responsiveness; the outcome of the insult depends upon the cellular developmental processes at a given embryonic stage.

Astrocytic involvement in learning and memory consolidation.

Astrocytes play fundamental roles in brain function, interacting with neurons and other astrocytes, yet their role in learning is not widely recognized. This review focuses on astrocytic involvement in memory consolidation following bead discrimination learning in day-old chick and draws parallels to mammalian learning, providing strong empirical support for the conclusion that the described neuronal-astrocytic interactions are universally valid. It identifies specific mechanisms whereby astrocytes support memory consolidation. Uptake of glucose, stimulated in astrocytes by beta(3)-noradrenergic receptor activation, provides energy by glycolytic/oxidative metabolism. Unlike neurons, astrocytes carry out net synthesis of tricarboxylic acid cycle intermediates needed for synthesis of transmitter glutamate formed by rapid degradation of glucose-derived glycogen and stimulated by beta(2)-noradrenergic receptor activation. This makes learning dependent on glycogenolysis and its stimulation by noradrenaline. Astrocytes take up most synaptically released glutamate, terminating transmitter activity and returning glutamate to neurons in a glutamate-glutamine cycle, interference with which abolishes learning. The various astrocytic activities follow a rigidly controlled time schedule, easily determined after bead discrimination learning but also detectable in other paradigms.

Inhibition of astrocytic energy metabolism by D-lactate exposure impairs memory.

Bead discrimination learning in day-old chicken was inhibited by bilateral injection into the intermediate medial mesopallium (IMM), a homolog of the mammalian brain cortex, of the poorly metabolized enantiomer of L-lactate, D-lactate. The window of vulnerability extended from 10 min before training to 20 min after training. Unilateral injection 10 min before training inhibited only in the left IMM, whereas 10 min after training injection was only inhibitory if made into the right hemisphere. The pre-training administration caused memory loss from the earliest time tested whereas memory was maintained for another 20 min when D-lactate was injected 10 min post-training. The ability of acetate, an astrocyte-specific substrate, injected into the IMM to counteract the inhibitory effect was tested. Following D-lactate injection 10 min before training, rescue of memory immediately after training was achieved by acetate as long as aspartate, an oxaloacetate precursor, was also present. This suggests that pyruvate carboxylation is necessary for net synthesis of glutamate, which is known to occur at this time [Gibbs, M.E., Lloyd, H.G.E., Santa, T., Hertz, L., 2007. Glycogen is a preferred glutamate precursor during learning in 1-day-old chick: biochemical and behavioral evidence. J. Neurosci. Res., 85, 3326-3333]. However, acetate alone rescued memory 20 min post-training (following d-lactate injection 10 min after training), indicating that pyruvate at this time is used for energy production, consistent with memory inhibition by dinitrophenol. These findings suggest that D-lactate acts by inhibiting uptake of L-lactate into astrocytes (an extracellular effect) or metabolism of pyruvate in astrocytic mitochondria (an intracellular effect). An apparent lag phase between the administration of d-lactate and its inhibition of learning favors the latter possibility. Thus, under the present experimental conditions D-lactate acts as an astrocytic metabolic inhibitor rather than as an inhibitor of neuronal L-lactate uptake, as has occasionally been suggested. Analogously, a rare reversible neurological syndrome with memory deficits, D-lactate encephalopathy, may mainly or exclusively be due to astrocytic malfunction.

The effect of hypoxia at different embryonic ages on impairment of memory ability in chicks.

Hypoxia during the prenatal period is a principal antecedent to cognitive impairment after birth. In this study we have investigated the duration, severity and timing of acute hypoxia during chick embryonic development to elucidate the relative importance of these factors. Our results show that 24h of hypoxia (exposure to 14% oxygen) at embryonic day 10 (E10) results in significant impairment of intermediate and long-term memory in the post-hatch chick, which is the same as we observed with 4 days of hypoxia. At E14, 24h of hypoxia, 5min of anoxia, but not 1h of hypoxia, resulted only in impaired long-term memory; the same as 4 days of hypoxia from E14. Corticosterone levels, measured post-hatch as an indicator of a stress response, were significantly elevated in response to E10 hypoxia, and E14 hypoxia (both 1 and 24h) and anoxia. In a separate experiment we exposed embryos to 24h of hypoxia from E6 to E16, and found that memory deficits resulted from hypoxia at E9 and E10, and E13-E15, while corticosterone concentrations at hatch were significantly raised following E10-E16 hypoxia. These results demonstrate that the developmental age when the insult occurs determines the nature of the cognitive deficit and, if the severity of the insult is sufficient, then the outcome, or deficits in memory ability, are consistent whether the insult is acute or chronic. Importantly, there are two critical stages in development, which in the chick are around E10 and E14, when acute hypoxia results in significant adverse cognitive effects after hatch. These time-points correspond to two different stages in growth and development.

The role of corticosterone in prehatch-induced memory deficits in chicks.

We have previously shown that prehatch hypoxia (14% oxygen for 24 h), at E10 or E14 of chick embryonic development, produces significant memory deficits, with E10 hypoxia significantly affecting short-term memory and the subsequent formation of long-term memory, whereas E14 hypoxia only affects long-term memory. One of the consequences of hypoxia is the release of stress hormones and we found in this study that hypoxia at E10 or E14 induced a significant increase in circulating corticosterone immediately after the cessation of hypoxia (E11 and E15, respectively). Corticosterone levels remained significantly elevated at hatch in the E14 hypoxia group. This study describes the effect of a single, in ovo, injection of corticosterone on subsequent memory ability in hatched chicks. It was found that corticosterone (0.2 nmol/egg) at E10 or E14 mimicked the memory deficits produced by hypoxia at the same prehatch ages. Embryos treated with corticosterone at E10 had poor short-term memory at hatch, whereas corticosterone administration at E14 resulted in poor long-term memory. Embryos treated with corticosterone at E16 had raised circulating corticosterone levels at hatch, but did not have impaired memory. Treatment with corticosterone at E10, E12, E14 and E16 produced the same cognitive outcomes as hypoxia at the same prehatch ages. However, elevated plasma corticosterone levels at hatch did not necessarily cause the impaired memory processing. Raised levels were observed after treatment at E14 when memory processing was impaired, at E16 when memory was not impaired and not at E10 when memory was impaired. This suggests that an acute rather than sustained increase in plasma corticosterone at particular developmental ages is the cause of impaired memory processing seen at hatch.

Role of adrenoceptor subtypes in memory consolidation.

Noradrenaline release in areas within the forebrain occurs following activation of noradrenergic cells in the locus coeruleus (LoC). Release of noradrenaline by attentional/arousal/vigilance factors appears to be essential for learning and is responsible for the consolidation of memory. Noradrenaline can activate any of nine different adrenoceptor (AR) subtypes in the brain and selectivity of action may be achieved by the spatial location and relative density of the AR subtypes, by different affinities of the different subtypes and by temporal selectivity in terms of when the different ARs are activated in the memory formation process. This review examines the use of selective agonists and antagonists to determine the roles of the AR subtypes in the one-trial discriminated avoidance learning paradigm in the chick. A model is developed that integrates noradrenergic activity in basal ganglia (lobus parolfactorius (LPO)) and association cortex (intermediate medial hyperstriatum ventrale (IMHV)) leading to the consolidation of memory 30 min after training. There is evidence that beta(2)- and beta(3)-ARs are important in the association area but require input from alpha(2)-AR stimulated activity in the basal ganglia for consolidation. On the other hand, alpha(1)-AR activation in the IMHV is inhibitory and prevents consolidation. While there is no role for beta(1)-ARs in memory consolidation, they play a role in short-term memory (STM). The use of the precocial chick has clear advantages in having a temporally discrete learning task which allows for discrimination memory and whose development can be followed at discrete intervals after learning. These studies reveal clear roles for AR subtypes in the formation and consolidation of memory in the chick, which have allowed the development of a model that can now be tested in mammalian systems.

Prenatal hypoxia impairs memory function but does not result in overt structural alterations in the postnatal chick brain.

We showed previously that hypoxia in ovo impairs memory consolidation in the chick tested 2 days after hatching. Our present aim was to investigate whether we could detect any morphological effects of the same prenatal hypoxia. Hypoxia was induced by half-wrapping the egg with an impermeable membrane from either days 10-18 (W10-18 chicks) or days 14-18 (W14-18 chicks) of incubation (hatching approximately 21 days). Measurement of blood gases showed that reducing the surface area of the egg for gas exchange resulted in reduced pO2 and increased pCO2 2 days after wrapping. Although this hypoxia was sufficient to impair cognitive processing in the postnatal chick, our data suggest that it did not produce overt structural alterations or changes in the number of neurons, glutamine synthetase-immunoreactive cells or immunoreactivity to synaptophysin in the presynaptic vesicles in the multimodal integration (cortical) area compared to controls. Hence, we found no differences in the astrocyte to neuron ratio, synaptic density and/or vesicle number. Analysis of the ontogeny of astrocytes during the prenatal period of hypoxia showed them to be present at embryonic day 12, but not at the earlier ages examined. Although we found cognitive deficits in chicks from embryos made hypoxic during incubation, our regimen of prenatal hypoxia did not alter any of the parameters measured in the brains. This does not preclude the possibility that changes have occurred at the cellular or molecular levels or in specific neurotransmitter systems.

Role of Glycogenolysis in Memory and Learning: Regulation by Noradrenaline, Serotonin and ATP.

This paper reviews the role played by glycogen breakdown (glycogenolysis) and glycogen re-synthesis in memory processing in two different chick brain regions, (1) the hippocampus and (2) the avian equivalent of the mammalian cortex, the intermediate medial mesopallium (IMM). Memory processing is regulated by the neuromodulators noradrenaline and serotonin soon after training glycogen breakdown and re-synthesis. In day-old domestic chicks, memory formation is dependent on the breakdown of glycogen (glycogenolysis) at three specific times during the first 60 min after learning (around 2.5, 30, and 55 min). The chicks learn to discriminate in a single trial between beads of two colors and tastes. Inhibition of glycogen breakdown by the inhibitor of glycogen phosphorylase 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) given at specific times prior to the formation of long-term memory prevents memory forming. Noradrenergic stimulation of cultured chicken astrocytes by a selective ß2-adrenergic (AR) agonist reduces glycogen levels and we believe that in vivo this triggers memory consolidation at the second stage of glycogenolysis. Serotonin acting at 5-HT2B receptors acts on the first stage, but not on the second. We have shown that noradrenaline, acting via post-synaptic α2-ARs, is also responsible for the synthesis of glycogen and our experiments suggest that there is a readily accessible labile pool of glycogen in astrocytes which is depleted within 10 min if glycogen synthesis is inhibited. Endogenous ATP promotion of memory consolidation at 2.5 and 30 min is also dependent on glycogen breakdown. ATP acts at P2Y1 receptors and the action of thrombin suggests that it causes the release of internal calcium ([Ca(2+)]i) in astrocytes. Glutamate and GABA, the primary neurotransmitters in the brain, cannot be synthesized in neurons de novo and neurons rely on astrocytic glutamate synthesis, requiring glycogenolysis.

Alpha 2-adrenoceptors in the basal ganglia have a role in memory consolidation and reinforcement.

This study demonstrates a role for alpha(2)-adrenoceptors in the basal ganglia in the consolidation of memory using weakly and strongly reinforced models of discriminated avoidance learning in the chick. The memory enhancing action of noradrenaline injected into the basal ganglia (lobus parolfactorius-LPO) was reduced in the presence of the alpha(2)-adrenoceptor antagonist yohimbine, but when noradrenaline was injected into the multi-modal association area (intermediate medial hyperstriatum ventrale-IMHV), yohimbine failed to prevent memory enhancement. Yohimbine injected into the LPO prevented, whereas the alpha(2)-adrenoceptor agonists oxymetazoline and clonidine enhanced, consolidation of memory. The timing of the inhibitory effect of yohimbine in the LPO suggested that alpha(2)-adrenoceptor involvement occurs 10-15 min after training, and that stimulation of alpha(2)-ARs in LPO is necessary for subsequent consolidation of memory. Oxymetazoline, being hydrophilic, was ineffective injected into IMHV, whereas the action of the lipophilic alpha(2)-adrenoceptor agonist clonidine in the IMHV was interpreted as an action at a site more distal in the brain, probably the LPO. The results suggest that noradrenaline release in the basal ganglia in the chick stimulates alpha(2)-adrenoceptors, which modulate and consolidate memory formation mediated by beta(2)- or beta(3)-ARs in the association area. The LPO may be responsible for the reinforcement of memory in the IMHV.

Hemispheric lateralization of memory stages for discriminated avoidance learning in the chick.

Memory formation following the one trial discriminated bead task in the chick falls into three stages (short-term, intermediate and long-term memory) that are defined by susceptibility to different classes of drugs. The stages show sharply timed offsets of sensitivity and loss at specific times after inhibition. Recall of the memory in the chick shows cyclical changes that differ in period between left and right hemispheres, and is marked by a series of brief windows of enhanced recall that recur with periods of about 16 and 25 min in the left and right hemispheres respectively. The timing of these 'retrieval events' corresponds, to a large extent, with the timing of the memory stages seen in the visual discrimination task. Here we examine the effects of left or right hemisphere injection of the main agents (glutamate, ouabain and anisomycin) that have been used to characterize the three stages of memory. We show that memory in the left hemisphere is largely responsible for performance at test and that processes involved in its consolidation generate the phases of memory.

Reciprocal changes in forebrain contents of glycogen and of glutamate/glutamine during early memory consolidation in the day-old chick.

Passive avoidance learning is with advantage studied in day-old chicks trained to distinguish between beads of two different colors, of which one at training was associated with aversive taste. During the first 30-min post-training, two periods of glutamate release occur in the forebrain. One period is immediately after the aversive experience, when glutamate release is confined to the left hemisphere. A second release, 30 min later, may be bilateral, perhaps with preponderance of the right hemisphere. The present study showed increased pool sizes of glutamate and glutamine, specifically in the left hemisphere, at the time when the first glutamate release occurs, indicating de novo synthesis of glutamate/glutamine from glucose or glycogen, which are the only possible substrates. Behavioral evidence that memory is extinguished by intracranial administration at this time of iodoacetate, an inhibitor of glycolysis and glycogenolysis, and that the extinction of memory is counteracted by injection of glutamine, supports this concept. A decrease in forebrain glycogen of similar magnitude and coinciding with the increase in glutamate and glutamine suggests that glycogen rather than glucose is the main source of newly synthesized glutamate/glutamine. The second activation of glutamatergic activity 30 min after training, when memory is consolidated into stable, long-term memory, is associated with a bilateral increase in pool size of glutamate/glutamine. No glycogenolysis was observed at this time, but again there is a temporal correlation with sensitivity to inhibition by iodoacetate and rescue by glutamine, indicating the importance of de novo synthesis of glutamate/glutamine from glucose or glycogen.

The effect of prenatal hypoxia and malnutrition on memory consolidation in the chick.

The contribution of hypoxia and malnutrition to cognitive impairments was investigated in chicks incubated in conditions of reduced gas exchange. Previous research has shown that reducing gas exchange during incubation by wrapping half the eggshell with an impermeable membrane results in impaired cognitive ability in young chicks. The results were interpreted within a three stage sequential model of memory using discriminated bead avoidance learning. Reducing gas exchange for 4 days from day 10 or 14, of the 21-day incubation, inhibits memory formation and consolidation into permanent storage. The nature of the cognitive deficit depended on the timing of the insult. Environmental hypoxia (14% oxygen), induced from days 10 to 14 and from days 14 to 18, replicated the memory deficits found previously when eggs were partially wrapped with a membrane. Oxygen is necessary to break down food and to provide energy to build tissue proteins, and therefore hypoxia (partial wrapping or environmental incubation) may indirectly cause malnutrition. Malnutrition, induced by removing 5%, 7.5% or 10% albumin from the egg prior to incubation, had no significant effect on memory consolidation. Raised corticosterone levels occurred in chicks malnourished by 5% and 7.5%, but brain sparing was only evident in chicks with 7.5% albumin removal. Hatch rates were very low in 10% malnourished chicks. Using the chick as a model of prenatal stress, we have been able to isolate the effects of hypoxia from contributing maternal factors.