The positive allosteric modulation of GABA A receptors mRNA in immature hippocampal rat neurons by midazolam affects receptor expression and induces apoptosis.

BACKGROUND: Numerous experimental studies show that anesthetics are potentially toxic to the immature brain. Even though benzodiazepines are widely used in pediatric anesthesia and intensive care medicine, only a few studies examine the effects of these drugs on immature neurons.

METHODS: Hippocampal neuronal cell cultures of embryonic Wistar rats (15 days in culture) were incubated with midazolam 100 or 300 nM for either 30 min or 4h. The time course of the mRNA expression of the glutamate receptors subunits NR1, NR2A and NR2B of the NMDA receptor, the GluA-1 and A-2 subunits of the AMPA receptor as well as the alpha 1 subunit of the GABAA receptor were examined by PCR. Apoptosis was detected using Western blot analysis for BAX, Bcl-2 and Caspase-3.

RESULTS: Midazolam at 100 and 300nM applied for 30 min and 100 nM for 4 h affected glutamate receptor and GABAA receptor subunit expression. However, these effects were reversible within 72 h following washout. When 300 nM midazolam was applied for 4 h a significant increase in the NR 1, and NR 2A mRNA subunit expression could be detected. The increase in NR 2B receptor subunit expression as well as the GluA1 subunit expression were not reversible within 72 h following washout. This increase in mRNA glutamate receptor subunit expression was associated with a significant increase in neuronal apoptosis.

CONCLUSION: In immature neurons midazolam altered GABA and glutamate mRNA receptor subunit expression. Prolonged increase in midazolam-induced glutamate receptor expression was associated with apoptosis.