Age-Dependency of Levetiracetam Effects on Exocytotic GABA Release from Nerve Terminals in the Hippocampus and Cortex in Norm and After Perinatal Hypoxia

Abstract

Perinatal hypoxia can lead to multiple chronic neurological deficits, e.g., mental retardation, behavioral abnormalities, and epilepsy. Levetiracetam (LEV), 2S-(2-oxo-1-pyrrolidiny1) butanamide, is an anticonvulsant drug with proven efficiency in treating patients with focal and generalized seizures. Rats were underwent hypoxia and seizures at the age of 10-12 postnatal days (pd). The ambient level and depolarization-induced exocytotic release of [³H]GABA (γ-aminobutyric acid) were analyzed in nerve terminals in the hippocampus and cortex during development at the age of pd 17-19 and pd 24-26 (infantile stage), pd 38-40 (puberty) and pd 66-73 (young adults) in norm and after perinatal hypoxia. LEV had no effects on the ambient [³H]GABA level. The latter increased during development and was further elevated after perinatal hypoxia in nerve terminals in the hippocampus during the whole period and in the cortex in young adults. Exocytotic [³H]GABA release from nerve terminals increased after perinatal hypoxia during development in the hippocampus and cortex, however this effect was preserved at all ages during blockage of GABA transporters by NO-711 in the hippocampus only. LEV realized its anticonvulsant effects at the presynaptic site through an increase in exocytotic release of GABA. LEV exerted more significant effect after perinatal hypoxia than in norm. Action of LEV was strongly age-dependent and can be registered in puberty and young adults, but the drug was inert at the infantile stage.

Keywords: Brain development; Cortex; Exocytosis; GABA; Hippocampus; Levetiracetam; Nerve terminals; Perinatal hypoxia.

Fig. 1

Experimental design

Fig. 2

Dose-response curve for the effect of LEV (10–250 µM) on 15 mM KCl-induced [³H]GABA release during blockage of GABA transporters by NO-711 at the age of pd 38–40

Fig. 3

15 mM KCl-induced [³H]GABA release from nerve terminals in the hippocampus without (a) and during blockage of GABA transporters by NO-711 (b) without LEV and in the presence of LEV (100 μM) at different postnatal period (pd 17–19, pd 24–26, pd 38–40 and pd 66–73) in norm (N) and after perinatal hypoxia (H). **p < 0.01 as compared to the norm pd 17–19 group; ⁺⁺p < 0.01 as compared to the hypoxia pd 17-19 group; ^§§p < 0.01 as compared to the norm pd 17–19 group in the presence of NO-711; ^#p < 0.05, ^##p < 0.01 as compared to the norm/hypoxia group without LEV of the appropriate age

Fig. 4

a Fluorescence emission spectra of acridine orange (AO) in the standard salt solution before and after application of LEV. b The absence of the acute effect of LEV on synaptic vesicle acidification. Synaptosomes were equilibrated with acridine orange (5 µM); when the steady level of the dye fluorescence was reached, LEV was added to synaptosomes. c Spike of acridine orange fluorescence in response to KCl-induced membrane depolarization after preliminary incubation of synaptosomes with LEV. d An increase in the fluorescence signal of acridine orange in response to application of 35 mM KCl in the control and after pre-incubation of synaptosomes with LEV. Data are mean ± SEM. **p < 0.01 as compared to the control

Fig. 5

a Fluorescence emission spectra of rhodamine 6G in the standard salt solution before and after application of LEV (100 µM). b The absence of the acute effect of LEV on the membrane potential of synaptosomes. The suspension of synaptosomes was equilibrated with potential-sensitive dye rhodamine 6G (0.5 µM); when the steady level of the dye fluorescence was reached, LEV (marked by arrow) was added to synaptosomes. Traces represent nine experiments performed with different preparations

Fig. 6

15 mM KCl-induced [³H]GABA release from nerve terminals in the cortex without (a) and during blockage of GABA transporters by NO-711 (b) without LEV and in the presence of LEV (100 μM) at different postnatal period (pd 17–19, pd 24–26, pd 38–40 and pd 66–73) in norm (N) and after perinatal hypoxia (H). **p < 0.01 as compared to the norm pd 17–19 group; ⁺⁺p < 0.01 as compared to the hypoxia pd 17–19 group; ^##p < 0.01 as compared to the norm/hypoxia group without LEV of the appropriate age

Fig. 7

The ambient level of [³H]GABA at different postnatal periods (pd 17–19, pd 24–26, pd 38–40 and pd 66–73) in the preparations of nerve terminals from the hippocampus (a) and from the cortex (b) in the presence of LEV (100 µM) in norm (N) and after perinatal hypoxia (H). **p < 0.01 as compared to the norm pd 17–19 group; ⁺⁺p < 0.01 as compared to the hypoxia pd 17–19 group; ^§p < 0.05; ^§§p < 0.01 as compared to the norm group of the appropriate age