Aggregation of the amyloid-beta (Abeta) peptide in the extracellular space of the brain is central to Alzheimer's disease pathogenesis. Abeta aggregation is concentration dependent and brain region specific. Utilizing in vivo microdialysis concurrently with field potential recordings, we demonstrate that Abeta levels in the brain interstitial fluid are dynamically and directly influenced by synaptic activity on a timescale of minutes to hours. Using an acute brain slice model, we show that the rapid effects of synaptic activity on Abeta levels are primarily related to synaptic vesicle exocytosis. These results suggest that synaptic activity may modulate a neurodegenerative disease process, in this case by influencing Abeta metabolism and ultimately region-specific Abeta deposition. The findings also have important implications for treatment development.

Aggregation of amyloid-beta (Abeta) peptide into soluble and insoluble forms within the brain extracellular space is central to the pathogenesis of Alzheimer's disease. Full-length amyloid precursor protein (APP) is endocytosed from the cell surface into endosomes where it is cleaved to produce Abeta. Abeta is subsequently released into the brain interstitial fluid (ISF). We hypothesized that synaptic transmission results in more APP endocytosis, thereby increasing Abeta generation and release into the ISF. We found that inhibition of clathrin-mediated endocytosis immediately lowers ISF Abeta levels in vivo. Two distinct methods that increased synaptic transmission resulted in an elevation of ISF Abeta levels. Inhibition of endocytosis, however, prevented the activity-dependent increase in Abeta. We estimate that approximately 70% of ISF Abeta arises from endocytosis-associated mechanisms, with the vast majority of this pool also dependent on synaptic activity. These findings have implications for AD pathogenesis and may provide insights into therapeutic intervention.

Extensive research has failed to clarify the mechanism of action of nitrous oxide (N2O, laughing gas), a widely used inhalational anesthetic and drug of abuse. Other general anesthetics are thought to act by one of two mechanisms-blockade of NMDA glutamate receptors or enhancement of GABAergic inhibition. Here we show that N2O, at anesthetically-relevant concentrations, inhibits both ionic currents and excitotoxic neurodegeneration mediated through NMDA receptors and, like other NMDA antagonists, produces neurotoxic side effects which can be prevented by drugs that enhance GABAergic inhibition. The favorable safety record of N2O may be explained by the low concentrations typically used and by the fact that it is usually used in combination with GABAergic anesthetics that counteract its neurotoxic potential.

Using high resolution capacitance measurements, we have characterized an ultrafast component of transmitter release in ribbon-type synaptic terminals of retinal bipolar neurons. During depolarization, capacitance increases to a plateau of approximately 30 fF with a time constant of approximately 1.5 ms. When not limited by activation kinetics of calcium current, the small pool is depleted even faster, with a time constant of 0.5 ms. After the ultrafast pool is depleted, capacitance rises with a slower time constant of approximately 300 ms. EGTA (5 mM) depresses the slower capacitance rise but leaves the ultrafast phase intact. BAPTA (5 mM) depresses both components of exocytosis. With paired-pulse stimulation, the ultrafast pool recovers from depletion with a time constant of approximately 4 s. The ultrafast component may represent fusion of docked vesicles at the base of the synaptic ribbon, while the slower component represents more distal vesicles on the ribbon.

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Although T-type calcium channels were first described in sensory neurons, their function in sensory processing remains unclear. In isolated rat sensory neurons, we show that redox agents modulate T currents but not other voltage- and ligand-gated channels thought to mediate pain sensitivity. Similarly, redox agents modulate currents through Ca(v)3.2 recombinant channels. When injected into peripheral receptive fields, reducing agents, including the endogenous amino acid L-cysteine, induce thermal hyperalgesia. This hyperalgesia is blocked by the oxidizing agent 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB) and the T channel antagonist mibefradil. DTNB alone and in combination with mibefradil induces thermal analgesia. Likewise, L-cysteine induces mechanical DTNB-sensitive hyperalgesia in peripheral receptive fields. These data strongly suggest a role for T channels in peripheral nociception. Redox sites on T channels in peripheral nociceptors could be important targets for agents that modify pain perception.

GABA A receptors are a pivotal inhibitory influence in the nervous system, and modulators of the GABA A receptor are important anesthetics, sedatives, anticonvulsants, and anxiolytics. Current views of receptor modulation suggest that many exogenous drugs access and bind to an extracellular receptor domain. Using novel synthetic steroid analogs, we examined the access route for neuroactive steroids, potent GABA A receptor modulators also produced endogenously. Tight-seal recordings, in which direct aqueous drug access to receptor was prevented, demonstrated that steroids can reach the receptor either through plasma membrane lateral diffusion or through intracellular routes. A fluorescent neuroactive steroid accumulated intracellularly, but recordings from excised patches indicated that the intracellular reservoir is not necessary for receptor modulation, although it can apparently equilibrate with the plasma membrane within seconds. A membrane impermeant neuroactive steroid modulated receptor activity only when applied to the inner membrane leaflet, demonstrating that the steroid does not access an extracellular modulatory site. Thus, neuroactive steroids do not require direct aqueous access to the receptor, and membrane accumulation is required for receptor modulation.

Although many glial cells possess neurotransmitter receptors and transporters, little is known about glial participation in neurotransmission. To explore this issue, we recorded neuronal autaptic and glial responses from cultured hippocampal single-neuron micro-islands. Excitatory synaptic events activate rapid electrogenic glial glutamate transporter currents similar to those elicited by exogenous glutamate in other preparations. We show here that glial transporter responses may be used to sense changes in presynaptic efficacy and that glial uptake helps to remove synaptically released glutamate, thereby contributing to the termination of excitatory synaptic currents under certain conditions. These observations provide a framework for understanding the role of glia in both normal and pathological processes.