Adenosine receptor activation is responsible for prolonged depression of synaptic transmission after spreading depolarization in brain slices.

Spreading depolarization (SD) is a slowly propagating, coordinated depolarization of brain tissue, which is followed by a transient (5-10min) depression of synaptic activity. The mechanisms for synaptic depression after SD are incompletely understood. We examined the relative contributions of action potential failure and adenosine receptor activation to the suppression of evoked synaptic activity in murine brain slices. Focal micro-injection of potassium chloride (KCl) was used to induce SD and synaptic potentials were evoked by electrical stimulation of Schaffer collateral inputs to hippocampal area Cornu Ammonis area 1 (CA1). SD was accompanied by loss of both presynaptic action potentials (as assessed from fiber volleys) and field excitatory postsynaptic potentials (fEPSPs). Fiber volleys recovered rapidly upon neutralization of the extracellular direct current (DC) potential, whereas fEPSPs underwent a secondary suppression phase lasting several minutes. Paired-pulse ratio was elevated during the secondary suppression period, consistent with a presynaptic mechanism of synaptic depression. A transient increase in extracellular adenosine concentration was detected during the period of secondary suppression. Antagonists of adenosine A1 receptors (8-cyclopentyl-1,3-dipropylxanthine [DPCPX] or 8-cyclopentyl-1,3-dimethylxanthine [8-CPT]) greatly accelerated fEPSP recovery and abolished increases in paired-pulse ratio normally observed after SD. The duration of fEPSP suppression was correlated with both the duration of the DC shift and the area of tissue depolarized, consistent with the model that adenosine accumulates in proportion to the metabolic burden of SD. These results suggest that in brain slices, the duration of the DC shift approximately defined the period of action potential failure, but the secondary depression of evoked responses was in large part due to endogenous adenosine accumulation after SD.

Binding of 7H-dibenzo[c,g]carbazole to polynucleotides and DNA in vitro.

The N-heterocyclic aromatic pollutant, 7H-dibenzo[c,g]carbazole (DBC), is a potent carcinogen having both local and systemic effects. The overall objective of this research was to investigate the nature of the covalent binding of DBC with nucleic acids in vitro. DBC was shown to bind to polynucleotides, RNA and DNA in an in vitro rat or hamster microsomal enzyme assay, exhibiting a preferential binding to polyguanylic acid (poly[G]). Benzo[a]pyrene (BaP) binding to these same nucleic acids was determined simultaneously and was approximately 10-fold higher than DBC binding under identical experimental conditions. DBC-nucleic acid binding was shown to be dependent upon the presence of a microsomal activating system, the results being similar for rat or hamster liver microsomes. This microsome-dependent binding was unaffected by the addition of epoxide hydrase activity modifiers but was almost completely inhibited by alpha-naphthoflavone. The nature of DBC-nucleic acid binding was investigated using fluorescence spectroscopy. Benzo[c]carbazole and 5,5,6,6-tetrahydrodibenzo[c,g]carbazole were synthesized as representatives of the effect of disruption of the DBC pi-electron system on fluorescence excitation and emission. DBC-poly[G] adducts were isolated from binding assay mixtures and separated by HPLC. Results indicated that there are at least three different DBC-poly[G] adducts formed in vitro. The emission spectra of isolated adducts were similar in shape to that of DBC; however, the adduct spectra were shifted 5-10 nm toward longer wavelengths. This suggests that the bound DBC species have intact pi-electron systems. Results are consistent with binding through the nitrogen position as well as binding through the 1,2,3,4-ring of the molecule.