Abstracts

Spontaneous, 4-AP induced seizures, as identified by paroxysmal electrical activity accompanied by a significant negative shift in extracellular voltage, occur after a significant period of high activity in the inhibitory cells. Seizures initiate as the inhibitory cells enter a transient state of depolarization block. The origin of this layer specific blockade of cellular spiking activity is unknown, but it shares physiologal similarity with hypoxic depolarization block. We present measurements with extremely high spatially resolutions utilizing nanoscale optical probes, PEBBLES (Barker et al, (1998) [italic]Anal. Chem[/italic]) as well as measurements from optically active oxygen sensing whole-cell mircoelectrodes., Two probes were used in these experiments. In the first case we have developed a technique to use a combination of electrical current and pressure to drive small nanoscale oxygen sensitive probes, or PEBBLES, into the extracellular space of the brain slices. In addition we coat the tip of a microelectrode with a mixture of fluorescent dyes which permit the measurement of local tissue oxygen pressure simultaneous with extracellular or intracellular electrical potential. A ratiometric dye cocktail is made by dissolving the following components, PVC - Poly(vinyl chloride), DOS - Bis (2-ethylhexyl) sebacate , PtOEPKt - Pt(II) Octaethylporphine Ketone and BODIPY 577/618 in the solvent THF [ndash] tetrahydrofuran. Borosilicate glass micropipettes (3-7 MW) are dipped in the above cocktail, calibrated and inserted into tissue. Light from a metal hallide light passes through a 580nm excitation filter, and is directed towards the sample using a 590 nm dichroic mirror. The fluorescence light is filtered alternatively at 620 nm and 760 nm to image the BODIPY and PtoEPKt-Pt(II) respectively., Intracellular electrical measurements reveal interleaving activity between excitatory cells in the st. pyramidale and inhibitory cells in the st. oriens. We recently presented simultaneous measurement of extracellular electrical activity and quantitative oxygen measurement on the level of microdomains within neural tissue. We have further refined this technique to measure simultaneous whole-cell electrical recordings with local oxygen measurements. Optical measurements using both small nanoscale PEBBLES as well as optodes display an increase in intensity during the seizure which corresponds to a decrease in the local oxygen pressure., We utilized nanoscale probes, PEBBLES, to measure the oxygen pressure across different strata in the CA1 region of the rat hippocampus during 4-AP induced seizures allowing a detailed investigation of the role that oxygen plays on the level of microdomains of cortical layers or even near single cells in the formation and termination of seizures., (Supported by F32 NS051072-01A1, K02MH01493, R01MH500006.)