Evidence group I mGluR drugs modulate the activation profile of lipopolysaccharide-exposed microglia in culture.

Metabotropic glutamate receptors (mGluRs) may play a role in modulating microglial activation, but group I mGluRs have received little attention. This study aimed to investigate the effects of group I mGluR selective ligands, (S)-3,5-dihydroxyphenylglycine ((S)-3,5-DHPG) and (RS)-1-aminoindan-1,5-dicarboxylic acid (AIDA), in lipopolysaccharide (LPS; 50 ng/ml)-activated rat microglial cultures. (S)-3,5-DHPG (150 microM) significantly reduced (approximately 20-60%) the LPS-mediated production of nitrite (NO2(-)), tumour necrosis factor alpha (TNF-alpha), and L-glutamate (Glu) at 24 and 72 h. Image analysis revealed increases in both cell area and number, with larger amoeboid microglia (with retracted processes) formed following 2 h LPS exposure. This cellular population was absent after addition of (S)-3,5-DHPG, an effect antagonised by AIDA, and a concomitant reduction in cell area was also found. Taken together, these biochemical and morphological observations suggest that (S)-3,5-DHPG reduces microglial activation, indicating a role for group I mGluRs in modulating microglial function.

Effects of lipopolysaccharide on glial phenotype and activity of glutamate transporters: Evidence for delayed up-regulation and redistribution of GLT-1.

Excitatory amino acid transporters (EAATs) are responsible for homeostasis of extracellular L-glutamate, and the glial transporters are functionally dominant. EAAT expression or function is altered in acute and chronic neurological conditions, but little is known about the regulation of EAATs in reactive astroglia found in such neuropathologies. These studies examined the effects of the bacterial endotoxin lipopolysaccharide (LPS) on glial EAATs in vitro. The effects of LPS (1 microg/ml, 24-72 h) on EAAT activity and expression were examined in primary cultures of mouse astrocytes. [(3)H]D-aspartate uptake increased to 129% of control by 72 h treatment with LPS. Saturation analysis revealed that apparent K(m) was unchanged whilst V(max) was significantly increased to 172% of control by 72 h LPS treatment. Biotinylation and Western blotting indicated that cell-surface expression of GLT-1 was significantly elevated (146% control) by LPS treatment whereas GLAST expression was unchanged. Confocal analyses revealed that LPS treatment resulted in cytoskeletal changes and stellation of astrocytes, with rearrangement of F-actin (as shown by phalloidin labelling). Immunocytochemistry revealed clustering of GLAST, and increased expression and redistribution of GLT-1 to the cell-surface following treatment with LPS. Similar experiments were conducted in microglia, where LPS (50 ng/ml) was found to up-regulate expression of GLT-1 at 24 and 72 h in concert with cytoskeletal changes accompanying activation. These findings suggest an association of cytoskeletal changes in glia with EAAT activity, with the predominant adaptation involving up-regulation and redistribution of GLT-1.

Establishment of primary cultures of rat olfactory bulb under serum-free conditions for studies of cellular injury.

Olfactory dysfunction has been implicated in various neurodegenerative diseases including Parkinson's and Alzheimer's disease but, despite intense interest in the neurobiology of the olfactory bulb (OB), studies of neurodegenerative mechanisms have not been attempted in primary OB cultures. This study was aimed at developing a primary OB culture under serum-free conditions in order to investigate injury and excitotoxicity in vitro. Olfactory bulbs from rat pups were rapidly trypsinised and mechanically dissociated and the resultant single cell suspension was centrifuged through a high bovine serum albumin concentration gradient to reduce cellular debris before being seeded in multi-well culture plates. Cells were plated in neurobasal medium containing 0.5 mM glutamine, 25 mM K(+), 2% B27 and 10% fetal calf serum (FCS) for 24 h and, after 1 day in vitro (div1), were maintained without FCS. At div8, neurones exhibited extensive neuritic networks, were present as a monolayer and were mainly bipolar and immunopositive for gamma-aminobutyric acid indicating that they were intrinsic OB neurones. At div8, neurones (positive for microtubule-associated protein-2, 73%) predominated over astrocytes (positive for glial fibrillary acidic protein, 27%). Cellular injury produced by staurosporine, hydrogen peroxide and kainate, when assessed by morphological and biochemical procedures, was shown to be concentration-dependent and significantly reduced the numbers of neurones and astrocytes. Further analyses of kainate-induced injury revealed the presence of TUNEL-positive cells (indicative of apoptosis) and increases in intracellular free calcium, both of which were antagonised by CNQX. Thus, the serum-free culture developed here is amenable to morphological and high throughput neurochemical analyses of mechanisms contributing to the injury of OB neurones in vitro.