Stephen R. Robinson

Neuronal–glial interactions and behaviour

Both neurons and glia interact dynamically to enable information processing and behaviour. They have had increasingly intimate, numerous and differentiated associations during brain evolution. Radial glia form a scaffold for neuronal developmental migration and astrocytes enable later synapse elimination. Functionally syncytial glial cells are depolarised by elevated potassium to generate slow potential shifts that are quantitatively related to arousal, levels of motivation and accompany learning. Potassium stimulates astrocytic glycogenolysis and neuronal oxidative metabolism, the former of which is necessary for passive avoidance learning in chicks. Neurons oxidatively metabolise lactate/pyruvate derived from astrocytic glycolysis as their major energy source, stimulated by elevated glutamate. In astrocytes, noradrenaline activates both glycogenolysis and oxidative metabolism. Neuronal glutamate depends crucially on the supply of astrocytically derived glutamine. Released glutamate depolarises astrocytes and their handling of potassium and induces waves of elevated intracellular calcium. Serotonin causes astrocytic hyperpolarisation. Astrocytes alter their physical relationships with neurons to regulate neuronal communication in the hypothalamus during lactation, parturition and dehydration and in response to steroid hormones. There is also structural plasticity of astrocytes during learning in cortex and cerebellum.

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.

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.

Chicks injected with antisera to either S-100α or S-100β protein develop amnesia for a passive avoidance task

The cellular expression of S-100 beta protein is upregulated in Alzheimers disease and in Downs 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.

Shifting relationships between photoreceptors and pigment epithelial cells in monkey retina: Implications for the development of retinal topography

This study examines the spatiotemporal relationships between retinal pigment epithelium (RPE) and photoreceptors (PR) during development of Macaca nemestrina retina. Our aim was to learn more about the developmental dynamics of these two important cell populations, particularly whether development changes in RPE cell densities mimic those of PR at selected retinal points. Twelve eyes ranging in age from 100 fetal days (Fd) to adulthood were flatmounted; the retinal perimeters were traced; and then sample punches were taken of the RPE and neural retina at the fovea, optic disc, mid- and far-nasal periphery, and far temporal, inferior and superior periphery. The two tissues were gently separated and the RPE cells and photoreceptors from the same region of the punch were counted using Nomarski contrast interference optics. We found that the total number of cones remains stable around 4 million between Fd100 and adulthood, but RPE number increases from 1.6 million at Fd100 to 2.56 million in adulthood. At the fovea, the core:RPE ratio increases from 5.4:1 at Fd100 to 28:1 by adulthood. In the temporal periphery by contrast, the cone:RPE ratio declines from 2.2:1 at Fd100-110 to less than 1:1 in the adult. In the vicinity of the optic disc, the ratio of (cones+rods); RPE remains around 35:1 throughout development, but in the retinal periphery it decreases to the adult value of 22:1. These changing ratios indicate that photoreceptors and RPE cells are redistributed independently during development, and that these two cellular sheets slide over one another to achieve their final distribution. This situation suggests that the forces or factors causing foveation are intrinsic to the neural retina.

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.

Relationships between Muller cells and neurons in a primitive tetrapod, the Australian lungfish

We recently proposed a model of cytogenesis which assumes that primitive ancestral mammals and premammalian vertebrates had a retinal composition that consisted of about seven neurons per Müller cell, comprising 1-2 cone photoreceptors, 1-2 rod photoreceptors, 2-3 bipolar cells, 1-2 amacrine cells, less than 1 ganglion cell, and less than 1 horizontal cell (Reichenbach & Robinson, 1995). The Australian lungfish (Neoceratodus forsteri) closely resembles the lobe-finned ancestors of land vertebrates, and has an extremely plesiomorphic nervous system. The present study, therefore, has examined the relative frequencies of retinal neurons and Müller cells (identified by immunolabelling for glutamine synthetase) in the lungfish retina. It was found that for each Müller cell there is an average of 1.9 cone photoreceptors, 1.7 rod photoreceptors, 3.1 amacrine/bipolar/horizontal cells, and 0.6 ganglion cells; amounting to a ratio of 7.3 neurons per Müller cell. These results support our conjecture that the sequence of cytogenesis in mammals is constrained by a developmental program that predates the evolution of mammals. The study also provides the first detailed morphological descriptions of lungfish Müller cells and their relationship with adjacent neurons. It was found that individual Müller cells in lungfish have a volume (more than 12,000 microns3) that is an order of magnitude higher than in mammals, yet the proportion of total retinal volume occupied by these cells (20%) is very similar.