R. K. P. Sullivan

Immunocytochemical analysis of D-serine distribution in the mammalian brain reveals novel anatomical compartmentalizations in glia and neurons

D‐Serine is a co‐agonist at the NMDA receptor glycine‐binding site. Early studies have emphasized a glial localization for D‐serine. However the nature of the glial cells has not been fully resolved, because previous D‐serine antibodies needed glutaraldehyde‐fixation, precluding co‐localization with fixation‐sensitive antigens. We have raised a new D‐serine antibody optimized for formaldehyde‐fixation. Light and electron microscopic observations indicated that D‐serine was concentrated into vesicle‐like compartments in astrocytes and radial glial cells, rather than being distributed uniformly in the cytoplasm. In aged animals, patches of cortex and hippocampus were devoid of immunolabeling for D‐serine, suggesting that impaired glial modulation of forebrain glutamatergic signaling might occur. Dual immunofluorescence labeling for glutamate and D‐serine revealed D‐serine in a subset of glutamatergic neurons, particularly in brainstem regions and in the olfactory bulbs. Microglia also contain D‐serine. We suggest that some D‐serine may be derived from the periphery. Collectively, our data suggest that the cellular compartmentation and distribution of D‐serine may be more complex and extensive than previously thought and may have significant implications for our understanding of the role of D‐serine in disease states including hypoxia and schizophrenia.

Glial glutamate transporter expression patterns in brains from multiple mammalian species

It is generally assumed that rodent brains can be used as representative models of neurochemical function in other species, such as humans. We have compared the distributions of the predominant glial glutamate transporters in rodents, rabbits, cats, pigs, monkeys, and humans. We identify similarities but also significant differences between species. GLT‐1v, which is abundantly expressed by rodent astrocytes, is expressed only in a rare subset of astrocytes of cats and humans, and appears to be absent from brains of rabbits and monkeys. Conversely, in the pig brain GLT‐1v is expressed only by oligodendrocytes. GLAST and GLT‐1α expression differed significantly between species; while rodents and rabbits exhibited uniform expression patterns in cortex, higher species, including cats, pigs, monkeys, and humans, exhibited heterogeneities in cortical and hippocampal expression. Patches devoid of labeling intermingling with patches of strong labeling were evident in areas such as temporal cortex and frontal cortex. In addition, we noted that in human motor cortex, there were inconsistencies in labeling for the C‐terminal of GLT‐1α and common domains of GLT‐1, suggesting that the C‐terminal region may be missing or that an unidentified splicing is present in many human astrocytes. Collectively our data suggest that assumptions as to the roles of glutamate transporters in any species may need to be tested empirically.

Cloning, Transport Properties, and Differential Localization of Two Splice Variants of GLT-1 in the Rat CNS: Implications for CNS Glutamate Homeostasis

At least two splice variants of GLT‐1 are expressed by rat brain astrocytes, albeit in different membrane domains. There is at present only limited data available as to the spatial relationship of such variants relative to the location of synapses and their functional properties. We have characterized the transport properties of GLT‐1v in a heterologous expression system and conclude that its transport properties are similar to those of the originally described form of GLT‐1, namely GLT‐1α. We demonstrate that GLT‐1α is localized to glial processes, some of which are interposed between multiple synapse types, including GABAergic synapses, whereas GLT‐1v is expressed by astrocytic processes, at sites not interposed between synapses. Both splice variants can be expressed by a single astrocyte, but such expression is not uniform over the surface of the astrocytes. Neither splice variant of GLT‐1 is evident in brain neurons, but both are abundantly expressed in some retinal neurons. We conclude that GLT‐1v may not be involved in shaping the kinetics of synaptic signaling in the brain, but may be critical in preventing spillover of glutamate between adjacent synapses, thereby regulating intersynaptic glutamatergic and GABAergic transmission. Furthermore, GLT‐1v may be crucial in ensuring that low levels of glutamate are maintained at extrasynaptic locations, especially in pathological conditions such as ischemia, motor neurone disease, and epilepsy.

Localization of taurine transporters, taurine, and 3H taurine accumulation in the rat retina, pituitary, and brain

The nervous system contains an abundance of taurine, a neuroactive sulfonic acid. Antibodies were generated against two cloned high‐affinity taurine transporters, referred to in this study as TAUT‐1 and TAUT‐2. The distribution of such was compared with the distribution of taurine in the rat brain, pituitary, and retina. The cellular pattern of [3H] taurine uptake in brain slices, pituitary slices, and retinas was examined by autoradiography. TAUT‐2 was predominantly associated with glial cells, including the Bergmann glial cells of the cerebellum and astrocytes in brain areas such as hippocampus. Low‐level labeling for TAUT‐2 was also observed in some neurones such as CA1 pyramidal cells. TAUT‐1 distribution was more limited; in the posterior pituitary TAUT‐1 was associated with the pituicytes but was absent from glial cells in the intermediate and anterior lobes. Conversely, in the brain TAUT‐1 was associated with cerebellar Purkinje cells and, in the retina, with photoreceptors and bipolar cells. Our data suggest that intracellular taurine levels in glial cells and neurons may be regulated in part by specific high‐affinity taurine transporters. The heterogeneous distribution of taurine and its transporters in the brain does not reconcile well with the possibility that taurine acts solely as a ubiquitous osmolyte in nervous tissues. GLIA 37:153–168, 2002.

Cytoskeletal Anchoring of GLAST Determines Susceptibility to Brain Damage AN IDENTIFIED ROLE FOR GFAP

Glial fibrillary acidic protein (GFAP) is an enigmatic protein; it currently has no unambiguously defined role. It is expressed in the cytoskeleton of astrocytes in the mammalian brain. We have used co-immunoprecipitation to identify in vivo binding partners for GFAP in the rat and pig brain. We demonstrate interactions between GFAP, the glutamate transporter GLAST, the PDZ-binding protein NHERF1, and ezrin. These interactions are physiologically relevant; we demonstrate in vitro that transport of d-aspartate (a glutamate analogue) is significantly increased in the presence of GFAP and NHERF1. Moreover, we demonstrate in vivo that expression of GFAP is essential in retaining GLAST in the plasma membranes of astrocytes after an hypoxic insult. These data indicate that the cytoskeleton of the astrocyte plays an important role in protecting the brain against glutamate-mediated excitotoxicity.

A new GLT1 splice variant: cloning and immunolocalization of GLT1c in the mammalian retina and brain

We have identified a novel carboxyl-terminal splice-variant of the glutamate transporter GLT1, which we denote as GLT1c. Within the rat brain only low levels of protein and message were detected, protein expression being restricted to end feet of astrocytes apposed to blood vessels or some astrocytes adjacent to the ventricles. Conversely, within the retina, this variant was selectively and heavily expressed in the synaptic terminals of both rod- and cone-photoreceptors in both humans and rats. Double-immunolabelling with antibodies to the carboxyl region of GLT1b/GLT1v, which is strongly expressed in apical dendrites of bipolar cells and in cone photoreceptors revealed that in the rat GLT1c was co-localised with GLT1b/GLT1v in cone photoreceptors but not with GLT1b/GLT1v in bipolar cells. GLT1c expression was developmentally regulated, only appearing at around postnatal day 7 in the rat retina, when photoreceptors first exhibit a dark current. Since the glutamate transporter EAAT5 is also expressed in terminals of rod photoreceptor terminals these data indicate that rod photoreceptors express two glutamate transporters with distinct properties. Similarly, cone photoreceptors express two glutamate transporters. We suggest that differential usage of these transporters by rod and cone photoreceptors may influence the kinetics of glutamate transmission by these neurons.

Immature Doublecortin-Positive Hippocampal Neurons Are Important for Learning But Not for Remembering

It is now widely accepted that hippocampal neurogenesis underpins critical cognitive functions, such as learning and memory. To assess the behavioral importance of adult-born neurons, we developed a novel knock-in mouse model that allowed us to specifically and reversibly ablate hippocampal neurons at an immature stage. In these mice, the diphtheria toxin receptor (DTR) is expressed under control of the doublecortin (DCX) promoter, which allows for specific ablation of immature DCX-expressing neurons after administration of diphtheria toxin while leaving the neural precursor pool intact. Using a spatially challenging behavioral test (a modified version of the active place avoidance test), we present direct evidence that immature DCX-expressing neurons are required for successful acquisition of spatial learning, as well as reversal learning, but are not necessary for the retrieval of stored long-term memories. Importantly, the observed learning deficits were rescued as newly generated immature neurons repopulated the granule cell layer upon termination of the toxin treatment. Repeat (or cyclic) depletion of immature neurons reinstated behavioral deficits if the mice were challenged with a novel task. Together, these findings highlight the potential of stimulating neurogenesis as a means to enhance learning.

Paradoxical enhancement of fear extinction memory and synaptic plasticity by inhibition of the histone acetyltransferase p300

It is well established that the coordinated regulation of activity-dependent gene expression by the histone acetyltransferase (HAT) family of transcriptional coactivators is crucial for the formation of contextual fear and spatial memory, and for hippocampal synaptic plasticity. However, no studies have examined the role of this epigenetic mechanism within the infralimbic prefrontal cortex (ILPFC), an area of the brain that is essential for the formation and consolidation of fear extinction memory. Here we report that a postextinction training infusion of a combined p300/CBP inhibitor (Lys-CoA-Tat), directly into the ILPFC, enhances fear extinction memory in mice. Our results also demonstrate that the HAT p300 is highly expressed within pyramidal neurons of the ILPFC and that the small-molecule p300-specific inhibitor (C646) infused into the ILPFC immediately after weak extinction training enhances the consolidation of fear extinction memory. C646 infused 6 h after extinction had no effect on fear extinction memory, nor did an immediate postextinction training infusion into the prelimbic prefrontal cortex. Consistent with the behavioral findings, inhibition of p300 activity within the ILPFC facilitated long-term potentiation (LTP) under stimulation conditions that do not evoke long-lasting LTP. These data suggest that one function of p300 activity within the ILPFC is to constrain synaptic plasticity, and that a reduction in the function of this HAT is required for the formation of fear extinction memory.

Distribution of two splice variants of the glutamate transporter GLT1 in the retinas of humans, monkeys, rabbits, rats, cats, and chickens

Antibodies have been generated against two carboxyl‐terminal splice variants of the glutamate transporter GLT1, namely, the originally described version of GLT1 and GLT1‐B, and labelling has been examined in multiple species, including chickens and humans. Although strong specific labelling was observed in each species, divergent patterns of expression were noted. Moreover, each antibody was sensitive to the phosphorylation state of the appropriate protein, because chemical removal of phosphates using alkaline phosphatase revealed a broader range of labelled elements in most cases. In general, GLT1‐B was present in cone photoreceptors and in rod and cone bipolar cells in the retinas of rabbits, rats, and cats. In the cone‐dominated retinas of chickens and in marmosets, GLT1‐B was associated only with cone photoreceptors, whereas, in macaque and human retinas, GLT1‐B was associated with bipolar cells and terminals of photoreceptors. In some species, such as cats, GLT‐B was also present in horizontal cells. By contrast, GLT1 distribution varied. GLT1 was associated with amacrine cells in chickens, rats, cats, and rabbits and with bipolar cells in marmosets and macaques. In the rat retina, rod photoreceptor terminals also contained GLT1, but this was evident only in enzymatically dephosphorylated tissues. We conclude that the two variants of GLT1 are present in all species examined but are differentially distributed in a species‐specific manner. Moreover, each cell type generally expresses only one splice variant of GLT1. J. Comp. Neurol. 445:1–12, 2002.

Distribution of two splice variants of the glutamate transporter GLT-1 in rat brain and pituitary

We have performed immunocytochemistry on rat brains using a highly specific antiserum directed against the originally described form of the glutamate transporter GLT‐1 (referred to hereafter as GLT‐1α), and another against a C‐terminal splice variant of this protein, GLT‐1B. Both forms of GLT‐1 were abundant in rat brain, especially in regions such as the hippocampus and cerebral cortex, and macroscopic examination of sections suggested that both forms were generally regionally coexistent. However, disparities were evident; GLT‐1α was present in the intermediate lobe of the pituitary gland, whereas GLT‐1B was absent. Similar marked disparities were also noted in the external capsule, where GLT1A labeling was abundant but GLT‐1B was only occasionally encountered. Conversely, GLT‐1B was more extensively distributed, relative to GLT‐1α, in areas such as the deep cerebellar nuclei. In most regions, such as the olfactory bulbs, both splice variants were present but differences were evident in their distribution. In cerebral cortex, patches were evident where GLT‐1B was absent, whereas no such patches were evident for GLT‐1α. At high resolution, other discrepancies were evident; double‐labeling of areas such as hippocampus indicated that the two splice variants may either be differentially expressed by closely apposed glial elements or that the two splice variants may be differentially targeted to distinct membrane domains of individual glial cells. GLIA 38:246–255, 2002.