Antiepileptic Drug Evaluation

The general approach to the study of antiepileptic substances in the slice has been one of inducing some quantifiable form of hyperexcitability, which is then followed by exposure of the slice to the drug in the perfusion fluid and additional quantification of the responses. For example, those using a typical slice method in the pharmaceutical industry first measure levels of neuronal discharge following exposure to penicillin and then again following 30 min of exposure to test compounds, quantitating any change in spike discharge rate as a measure of potential antiepileptic activity.24

a. ACSF Controls

Since evaluation of drug exposure in the slice may require prolonged perfusion and time for subsequent data collection periods, it is essential that any changes in function of the slice over time in the absence of any treatment whatsoever be evaluated. Any decrements of function so quantified should be taken into consideration during interpretation of results obtained with other treatments added to the protocol.

b. Methods of Exposure

Since the experimental apparatus requires a constant flow of ACSF over the slice, it is easy to add convulsant or anticonvulsant chemicals to the perfusion fluid for a predetermined length of time. We attach two 50 cc plastic syringes to a common manifold and turn one supply off (e.g., ACSF + convulsant) while quickly turning on the other (e.g., ACSF + convulsant + anticonvulsant). Figure 10.6 shows this simple arrangement for supplying the perfusion fluids during an experiment. This method of treatment enables one to vary the level of exposure (dose), but requires prolonged perfusion (30 min or more) with most agents to guarantee some level of equilibration with the slices themselves.

Alternatively, known concentrations of agents could be added directly to the slices a drop at a time in the perfused chamber, but this method would have the disadvantage of having variable and rapidly changing drug concentrations with the agent being flushed away fairly quickly at normal perfusion rates. Ionophoretic application of test substances has also been employed.25

c. Vehicles

The choice of a solvent for dissolving the chemical under study may be problematic unless the agent dissolves easily in aqueous solvents. Agents such as DMSO (Merck, Rahway, NJ) or surface active agents such as tweens or spans (Aldrich Chemical, Milwaukee, WI) or even alcohol may be required to get the agent into solution, only to have it precipitate back out when it reaches the ACSF perfusing the slice. Appropriate control experiments must be performed examining the effects of all vehicles before interpreting any experimental findings following their use.

MANIFOLD FOR DELIVERY OF PERFUSION FLUIDS

A= ACSF WITH CONVULSANT

B= ACSF, CONVULSANT AND TEST ANTICONVULSANT

FIGURE 10.6

Side view of two syringes (50 cc each) mounted on a common manifold through which perfusing fluids reach slices in the recording chamber. Control experiments are done with artificial cerebrospinal fluid (ACSF) alone. The fluid from chamber A then perfuses the slices for 30 min in order to induce hyperexcitability (epileptiform events) while additional measurements are made. Finally, the solution from chamber B is used to test for anticonvulsant actions produced by the test substance. Note that the convulsant continues to be included in the solution while evaluating the test substance.

A= ACSF WITH CONVULSANT

B= ACSF, CONVULSANT AND TEST ANTICONVULSANT

FIGURE 10.6

Side view of two syringes (50 cc each) mounted on a common manifold through which perfusing fluids reach slices in the recording chamber. Control experiments are done with artificial cerebrospinal fluid (ACSF) alone. The fluid from chamber A then perfuses the slices for 30 min in order to induce hyperexcitability (epileptiform events) while additional measurements are made. Finally, the solution from chamber B is used to test for anticonvulsant actions produced by the test substance. Note that the convulsant continues to be included in the solution while evaluating the test substance.

d. End Points for Evaluation

Antiepileptic effects of drug treatments may be evaluated based on any of the chosen end points or measurements discussed. Any distortions or changes in baseline measures induced by convulsants, for example, can be altered back towards normal values by anticonvulsant drug exposure in the slice.15 Restoration of levels of feedback inhibition after exposure to anticonvulsants may also indicate some potentially useful antiepileptogenic properties of the test agent.15 Drug treatment may diminish levels of hyperexcitability, i.e., decreasing the number of multiple population spikes following exposure to convulsants.15

e. Reevaluation or Time Course

We have measured the time course of drug effects in the hippocampal slice by reevaluating all measures at 10, 30, and 60 min after continuous drug exposure via the perfusion fluid. Full interpretation of data collected from such a protocol would again require similar studies using only the vehicle for the same treatment period.

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