Figure 4.

The 5-selective compound SH-053-2′F-R-CH3 enhances neuronal activity in cortical slice cultures during up states.

The upper part of the figure displays the mean frequency of action potential firing during up states under control condition (black) and in the presence of the benzodiazepine (grey). All up states were collected, divided into time bins of 10 ms and averaged. The graph shows the mean value of action potential frequency, measures of statistical dispersion have been omitted for clarity reasons. In the lower part of the figure the difference between drug condition divided by control condition is shown to illustrate the effect of the drug on action potential firing during up state.

(A) SH-053-2′F-R-CH3 at a concentration of 10μM (n = 9) leads to an increase in action potential frequency during up states in wild type slices (grey line, marked by the arrow). Although SH-053-2′F-R-CH3 is a positive modulator at GABA_A receptors, the compound does not depress, but instead increases neuronal activity during up states. This increase in action potential frequency is the more pronounced the longer the up state lasts (activity approximately twice as high compared to control condition from 300ms to 500ms after the beginning of the up state), as indicated in the lower part of the figure.

(B) In cultured slices from the 5(H105R) mutant the increase in action potential frequency during up states by SH-053-2′F-R-CH3 (10μM, n = 10) is absent. This is consistent with this effect being mediated by 5-containing GABA_A receptors.

(C) L 655,708 acts as an inverse agonist at the benzodiazepine site of 5-containing GABA_A receptors and can thereby be regarded as an “anti- SH-053-2′F-R-CH3”. In fact, the neuronal activity of cultured cortical neurons shows nearly opposing behaviour in the presence of 2.5 μM L 655,708 compared to SH-053-2′F-R-CH3. The depression of neuronal activity is rather uniform beginning around 50 ms after the onset of the activity phase.

(D) The classical benzodiazepine diazepam (12.5μM, n = 13) leads to a depression of action potential frequency with a maximum around 50 ms after the beginning of the up state (marked by arrowhead in the upper part) in cultured slices from wild type mice. Note the small but steady relative increase in activity, starting at 40ms and reaching control levels at 200ms after up state onset is evident. This upward-sloping course of action potentials points to a similar, activity-enhancing action of diazepam which is superimposed by its overall depression of the cortical network.