L. T. Macnab

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.

GLAST1b, the exon-9 skipping form of the glutamate-aspartate transporter EAAT1 is a sensitive marker of neuronal dysfunction in the hypoxic brain

In normal brain, we previously demonstrated that the exon-9 skipping form of glutamate-aspartate transporter (GLAST; which we refer to as GLAST1b) is expressed by small populations of neurons that appear to be sick or dying and suggested that these cells were subject to inappropriate local glutamate-mediated excitation. To test this hypothesis we examined the expression of GLAST1b in the hypoxic pig brain. In this model glial glutamate transporters such as GLAST and glutamate transporter 1 (GLT-1) are down-regulated in susceptible regions, leading to regional loss of glutamate homeostasis and thus to brain damage. We demonstrate by immunohistochemistry that in those brain regions where astroglial glutamate transporters are lost, GLAST1b expression is induced in populations of neurons and to a lesser extent in some astrocytes. These neurons were also immunolabeled by antibodies against the carboxyl-terminal region of GLAST but did not label with antibodies directed against the amino-terminal region. Our Western blotting data indicate that GLAST1b expressed by neurons lacks the normal GLAST amino-terminal region and may be further cleaved to a smaller approximately 30-kDa fragment. We propose that GLAST1b represents a novel and sensitive marker for the detection of neurons at risk of dying in response to hypoxic and other excitotoxic insults and may have wider applicability in experimental and clinical contexts.

Expression of the exon 9–skipping form of EAAT2 in astrocytes of rats

mRNA for the exon 9-skipping form of the glutamate transporter excitatory amino acid transporter (EAAT) 2 (glutamate transporter 1, GLT-1) is known to be expressed in brain and spinal cord, and such expression was initially proposed to be associated with motor neuron disease. Surprisingly, a protein corresponding to the size of this splice variant has not previously been detected when using antibodies against one of the possible carboxyl terminal regions of EAAT2. This has been construed as indicating that little of the exon 9-skipping protein is expressed, or that such protein is not stable. We have now made selective antibodies against the splice site of this form of EAAT2. We show that in the adult rat brain and spinal cord, it is expressed primarily in populations of white matter astrocytes. Astrocytes expressing this splice variant also expressed glial fibrillary acidic protein. Expression was developmentally regulated, being expressed in a small number of astrocytes at postnatal day 7, but strongly expressed by large populations of white matter astrocytes by 25 days postnatum and into adulthood. Only a subset of gray matter astrocytes and radial glia expressed exon 9-skipping EAAT2. We suggest that exon 9-skipping EAAT2 may have a role in regulating extracellular glutamate in white matter tracts, either by interacting with normally spliced EAAT2 and modifying its targeting or transport activity, or by acting as a transporter itself. Conversely, the limited expression in gray matter suggests it is unlikely to be important for modulating synaptic levels of glutamate.

Expression of the exon 3 skipping form of GLAST, GLAST1a, in brain and retina

GLAST is a glial glutamate transporter; mRNA for a splice variant, GLAST1a, which lacks exon 3, has previously been identified. To detect GLAST1a protein, we generated antibodies against a peptide sequence encompassing the splice site. We demonstrate by Western blotting and immunocytochemistry the expression of GLAST1a in brains and retinae. Robust immunolabelling was present in the cerebellar Bergmann glia, and weaker labelling was evident in the retinal Müller cells. GLAST1a is differentially targeted to some cellular compartments such as the end feet of the Müller cells. As GLAST1a protein may interfere with the transport of glutamate by normally spliced GLAST, differentially targeted expression of GLAST1a may represent a mechanism for selectively regulating GLAST function in the mammalian nervous system.

Central nervous system expression of the exon 9 skipping form of the glutamate transporter GLAST.

We have raised antibodies that selectively recognize an exon 9 skipping form of GLAST. We demonstrate expression of this protein in brains of rats, cats, monkeys and humans. Immunolabelling was present in scattered populations of neurons, particularly in cerebral cortex and colliculi. Neurons were often present in small clusters and exhibited a range of morphologies, from apparently normal to highly degenerate. GLAST1b was also expressed by some glial cells. Cortical neurons expressing the exon 9 skipping form of GLAST also labelled with antibodies against the C- or N-terminal regions of GLAST. We suggest that alternate splicing of GLAST by subpopulations of neurons may indicate some dysfunction in these cells, and may be an indicator of inappropriate local excitation.

Cryptic expression of functional glutamate transporters in the developing rodent brain.

The co-ordinate functioning of neurons and glia is required for glutamate-mediated neurotransmission. In this study, we show by immunocytochemical detection of D-aspartate uptake, that functional glutamate transporters are present in the developing CNS of fetal and neonatal rats, including forebrain, midbrain and hindbrain, at least as early as embryonic day 12 (E12). Use of the transport inhibitor dihydrokainic acid revealed a significant role for GLT-1 in the uptake process.Immunolabelling for the glutamate transporters GLAST, GLT-1alpha and GLT-1v showed that each of these proteins are expressed early in development and appear to be restricted to glial-like cells throughout the development period examined (except in the retina, where neuronal elements were also labelled). Our capacity to detect very early expression of the variant forms of GLT-1 contrasts with other studies, a feature that we attribute to the use of antigen-recovery techniques that unmask protein epitopes that are otherwise undetectable. These studies illustrate the widespread presence of functional glutamate transporters in the developing CNS, in many cases before the onset of periods of synaptogenesis and indicate that regulation of extracellular glutamate by multiple excitatory amino acid transporters might be crucial in early CNS development.