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Growing numbers of studies indicate that polysaccharides influence signaling events important for brain function. It has been speculated that such polysaccharide modulation of neuronal signals can promote synaptogenesis and cell maintenance. Here, we tested whether dextran sulfate, a polyanion that mimics natural mucopolysaccharides, protects hippocampal neurons against excitotoxic insults. An excitotoxin was applied to primary hippocampal cultures in the absence or presence of a large 500-kDa dextran sulfate (DS-L), a smaller 5-8-kDa species (DS-S), or sulfate-free dextran of 500 kDa. Only DS-L prevented neuronal damage as determined by a membrane permeability assay and phase contrast morphology. The sulfate and size dependence is also characteristic of DS-L's modulatory action on the channel activity of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type glutamate receptors. The extent of neuroprotection correlates with the level of modulation of AMPA responses, and DS-L exhibits comparable EC(50) values for the two effects (3-7 nM). DS-L also modulates the link between AMPA receptors and mitogen-activated protein kinase (MAPK) involving extracellular signal-regulated protein kinase (ERK), well known for its involvement in cell survival and repair. Correspondingly, protection against N-methyl-D-aspartate (NMDA) excitotoxicity was evident in hippocampal slice cultures when DS-L was applied 30 min postinsult. These findings suggest that polysaccharides elicit neuroprotection in the brain, including enhanced repair responses through the AMPA receptor-MAPK axis.

Soman, an anticholinesterase and dangerous nerve agent, produces convulsions, memory impairment, and cell loss in the brain, especially in the hippocampus. Soman-induced accumulation of acetylcholine initiates mechanisms responsible for the development of incapacitating seizures. The prolonged epileptiform nature of these seizures causes the release of another excitatory neurotransmitter, glutamate, which has been linked to the toxic action of the nerve agent. Here, we tested whether subtoxic soman exposures influence the brain's sensitivity to glutamate-based excitotoxicity. Over a 1-week period, hippocampal slice cultures were exposed daily to a transient level of soman that produced no evidence of synaptic deterioration. After the subtoxic soman treatments, however, the tissue became vulnerable to a brief episode of glutamate receptor overstimulation that normally resulted in little or no excitotoxic damage. In those slice cultures treated with subtoxic soman, a decline in synaptic markers as well as an increase in spectrin breakdown occurred 24 hr after the mild excitotoxic event. Exposure to high soman concentrations alone produced similar synaptic degeneration, but without evident cell death, suggesting that synaptic decline is an early neurotoxicological response to the nerve agent. Interestingly, enhanced excitotoxic sensitivity caused the brain tissue to become susceptible to disparate insults initiated before or after the soman contact. These findings indicate that seemingly innocuous soman exposures leave the hippocampus sensitive to the types of insults implicated in traumatic brain injury and stroke. They also warn that asymptomatic contact with soman may lead to progressive synaptopathogenesis and that early indicators of soman exposure are critical to prevent potential brain injury.

Previous work found evidence that alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-type glutamate receptors interact with and are functionally regulated by the glycosaminoglycan heparin. The present study tested whether dextran species affect ligand binding, channel kinetics, and calcium permeability of AMPA receptors. Dextran sulfate of 500 kDa markedly reduced high affinity [3H]AMPA binding in solubilized hippocampal membranes. In isolated receptors reconstituted in a lipid bilayer, the same dextran sulfate prolonged the lifetime of open states exhibited by AMPA-induced channel fluctuations. The large polysaccharide further changed the single channel kinetics by increasing the open channel probability five- to sixfold. Such modulation of channel activity corresponded with enhanced levels of calcium influx as shown in hippocampal neurons loaded with Fluo3AM dye. With an exposure time of <1 min, AMPA produced a dose-dependent increase in intracellular calcium that was blocked by 6-cyano-7-nitroquinoxaline-2,3-dione disodium (CNQX). Dextran sulfate, at the same concentration range that modified ligand binding (EC50 of 5-10 nM), enhanced the AMPA-induced calcium influx by as much as 60%. The enhanced influx was blocked by CNQX, although unchanged by the N-methyl-D-aspartate (NMDA) receptor antagonist AP5. Confocal microscopy showed that the increase in calcium occurred in neuronal cell bodies and their processes. Interestingly, smaller 5-8-kDa dextran sulfate and a non-sulfated dextran of 500 kDa had little or no effect on the binding, channel, and calcium permeability properties. Together, these findings suggest that synaptic polysaccharide species modulate hippocampal AMPA receptors in a sulfate- and size-dependent manner., (Copyright 2003 Wiley-Liss, Inc.)

The cysteine protease calpain is activated by calcium and has a wide range of substrates. Calpain-mediated cellular damage is associated with many neuropathologies, and calpain also plays a role in signal transduction events that are essential for cell maintenance, including the activation of important kinases and transcription factors. In the present study, the hippocampal slice culture was used as a model of excitotoxicity to test whether the neuroprotection elicited by selective calpain inhibition is associated with changes in cell signaling. Peptidyl alpha-keto amide and alpha-keto acid inhibitors reduced both calpain-mediated cytoskeletal damage and the concomitant synaptic deterioration resulting from an N-methyl-D-aspartate exposure. The alpha-keto amide CX295 was protective when infused into slice cultures before or after the excitotoxic episode. The slices protected with CX295 exhibited normal activation levels of mitogen-activated protein kinase and the transcription factor nuclear factor-kappaB. Thus, selective inhibition of calpain provides neuroprotection without influencing critical signaling pathways.

Adhesion responses triggered by integrin-class matrix receptors have been implicated in the synaptic reorganization events necessary for certain types of neuronal plasticity. Hippocampal slice cultures were used to test whether the related structural transformations elicited by NMDA receptor stimulation are regulated by integrin-type signals. Infusing the slices with NMDA for a short period induced the expected disassembly of the cytoskeletal network, measured with antibodies that selectively recognize spectrin cleavage sites targeted by the protease calpain. Marked levels of the 150-kDa breakdown product (BDP) were produced, whereas concentrations of the parent spectrin were not changed. Interestingly, the calpain cleavage events were attenuated by 60% when integrin-type signaling was disrupted with the antagonist Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP). This effect was RGDS-dependent, was largely evident in synapse-dense dendritic areas, particularly in subfield CA1, and was abolished when the NMDA exposure period was >5 min. These findings suggest that only those cytoskeletal alterations associated with brief synaptic activity are regulated by intact contact zones. AMPA-type glutamate receptors also were tested because, like spectrin, they are targets for calpain. Brief NMDA treatment caused a 15% loss of AMPA receptor GluR1 carboxytermini and this modification was augmented to 32% in the presence of GRGDSP. Thus, although blockage of matrix recognition signals decreased spectrin's susceptibility to disassembly, it increased the susceptibility of AMPA receptors to proteolysis. These data indicate that integrin-type signaling complexes are appropriately positioned to govern cytoskeletal reconfiguration while stabilizing the structural nature of AMPA receptors., (Copyright 2000 Wiley-Liss, Inc.)

The postsynaptic molecule gephyrin is involved in clustering neurotransmitter receptors. To test for protein variants that correspond to alternatively spliced gephyrin mRNAs, antibodies were made against 1) an amino-terminal domain of gephyrin (GN(N)) and 2) its invariant carboxy-terminus (GN(C)). Both antibodies recognized an antigen with the expected molecular weight of 93-95 kDa in rat and human brain tissue, as well as five additional proteins between 90 and 108 kDa. Most of these variants were found distributed throughout the brain, and their developmental profiles paralleled those of synaptic markers. Interestingly, the pattern of antigens immunostained across brain regions by anti-GN(N) was markedly distinct from that labeled by anti-GN(C), a difference consistent with carboxy-terminal modification. In control experiments in which hippocampal membranes were treated to activate endogenous proteases, there was no evidence that certain gephyrin variants originate from proteolysis. A subset of the antigens was, however, rapidly degraded during the treatment. A corresponding production of stable, carboxy-terminal gephyrin fragments of 48-50 kDa occurred within 1 min of proteolytic activation and was blocked by the selective calpain inhibitor CX295. These findings suggest that multiple gephyrin proteins are active in the brain and that some of their roles may require functional modulation by limited proteolysis.

Organotypic cultures provide a unique strategy with which to examine many aspects of brain physiology and pathology. Long-term slice cultures from the hippocampus, a region involved in memory encoding and one that exhibits early degeneration in Alzheimer's disease and ischemia, are particularly valuable in this regard due to their expression of synaptic plasticity mechanisms (e.g., long-term potentiation) and responsiveness to pathological insults (e.g., excitotoxicity). Long-term slices can be prepared from hippocampi at the second or third postnatal week of development and thus incorporate a number of relatively mature features; further signs of maturation and the preservation of adult-like characteristics occur over succeeding weeks. The stability of the cultured slice renders it an appropriate model for studying 1) prolonged regulation/stabilization events linked to synaptogenesis and certain forms of plasticity, 2) temporal patterns of cellular atrophy associated with pathogenic conditions such as ischemia and epilepsia, and 3) slow processes associated with aging and age-related pathologies.