Effects pf hypoxia and antiepileptic drugs of electrophysiological properties of CA1 neurons in hippocampus

This thesis is based on three experimental studies (I-III) of CA1 hippocampal neurons in rat brain slices, where the electrophysiological properties are examined with patch clamp technique under hypoxic conditions and during treatment with antiepileptic drugs, respectively. The cells were visualized and identified in the slice by means of infrared differential interference contrast (IR-DIC) video microscopy and the experiments were carried out at room temperature (22-25 ºC). The major part of the analysis was performed with whole-cell current-clamp technique in order to study the effects on the cell potential. A voltage-clamp analysis on cell-attached patches was included in study II.

Study I: The effect of chemical hypoxia (cyanide) was studied in order to elucidate the reason for previously found differences in the reaction to hypoxia in CA1 cells. This study focused on the changes in resting membrane potential. Chemical hypoxia was found to change the membrane potential with a magnitude and direction depending on the original resting potential of the cells. Several findings suggested that chemical hypoxia activated not only K channels, but in addition increased a conductance for an ion with a more positive equilibrium potential, most likely to be the Na+ ion. Blockage of the active transport (the Na/K pump) with ouabain potentiated the depolarizing action of cyanide, but did not inhibit the post-cyanide (“post-hypoxic”) hyperpolarization indicating that this part of the effect is not dependent on a reactivation of the Na/K pump.

Study II: The objective was to distinguish if the rapid changes in excitability during hypoxia are related to a decrease in the Na conductance or if they are secondary to changes in the membrane potential. Hypoxia was induced by cyanide as in study I. Cyanide caused a decrease in the excitability measurable as an increase in the current threshold for excitation, which was correlated to a change in the resting potential. There were no effects on the action potential properties, such as rate of rise or peak amplitude. In conclusion, the study showed that there was no decrease in Na conductance in CA1 cells during chemical hypoxia, and that the rapid hypoxic effects on excitation can be attributed to the membrane potential changes.

Study III: The mode of action of the commonly used antiepileptic drugs VPA, LTG and LEV has not been fully clarified, and this was the rationale for this study. The experiments intended to study the rapid effects on excitability and firing properties as seen during drug perfusion for 10 min. All effects were compared with those in control cells in order to distinguish the normal changes over time in perfused CA1 cells. The excitability, the action potential shape and amplitude, and the firing properties of CA1 cells were affected by LTG consistent with a block of Na channels, which may explain its antiepileptic mode of action. VPA and LEV did not show these effects and the mechanism of their antiepileptic action need to be different. Our findings differ in some respects from reported effects of VPA and LEV in cultured or dissociated neurons, and it is likely that the effects in the hippocampal slice, where the cells are relatively intact, are more similar to the therapeutic action.

List of scientific papers

I. Englund M, Hyllienmark L, Brismar T (2001). Chemical hypoxia in hippocampal pyramidal cells affects membrane potential differentially depending on resting potential. Neuroscience. 106(1): 89-94
https://pubmed.ncbi.nlm.nih.gov/11564419

II. Englund M, Bjurling M, Edin F, Hyllienmark L, Brismar T (2004). Hypoxic excitability changes and sodium currents in hippocampus CA1 neurons. Cell Mol Neurobiol. 24(5): 685-94
https://pubmed.ncbi.nlm.nih.gov/15485138

III. Englund M, Hyllienmark L, Brismar T (2007). Effect of valproate, lamotrigine and levetiracetam on excitability and firing properties of CA1 neurons in rat brains slices. [Submitted]