The flavonoid acetylpectolinarin counteracts the effects of low ethanol on spontaneous network activity in hippocampal cultures.

ETHNOPHARMACOLOGICAL RELEVANCE: A major concern in modern society involves the lasting detrimental behavioral effects of exposure to alcoholic beverages. Consequently, hundreds of folk remedies for hangover have been suggested, most of them without a scientific basis, for lack of proper test systems. Over centuries, yellow toadflax (Linaria vulgaris Mill., Lv) tincture has been used in Russian traditional medicine to treat the spectrum of hangover symptoms such as vertigo, headache, drunken behaviors, and as a sedative. MATERIALS AND METHODS: Here we use in-vitro cultured hippocampal neurons to examine the effect of the Lv extract as well as the flavonoid acetylpectolinarin (ACP) exclusively found in Lv extract, on spontaneous network activity of the cultured neurons exposed to low, physiological concentrations of ethanol. RESULTS: As in previous studies, low (0.25-0.5%) ethanol causes an increase in network activity, which was converted to suppression, with high concentrations of ethanol. Lv extract and ACP, at low concentrations, had no appreciable effect on spontaneous activity, but they blocked the facilitating action of low ethanol. This action of ACP was also seen when the culture was exposed to 1-EBIO, a SK potassium channel opener, and was blocked by apamin, an SK channel antagonist. In contrast, ACP or Lv extracts did not reverse the suppressive effects of higher ethanol. CONCLUSIONS: Our results suggest that ACP acts by interacting with the SK channel, to block the facilitatory effect of low concentration of ethanol, on network activity in hippocampal cultures.

Complex effects of aqueous extract of Melampyrum pratense and of its flavonoids on activity of primary cultured hippocampal neurons.

ETHNOPHARMACOLOGICAL RELEVANCE: The aqueous extract of the plant Malmpyrum pratense (Mp), is widely used in traditional medicine as a sedative, yet the biological basis of its action is not known. AIM OF THE STUDY: The effects of Mp on network activity and intrinsic and synaptic properties were studied in cultured hippocampal neurons in an attempt to analyze its mode of action. MATERIALS AND METHODS: Dissociated cultures of rat hippocampal neurons were used. Spontaneous network activity was assessed by variations in intracellular [Ca(2+)] concentrations, reflecting action potential discharges. Individual neuronal synaptic activity was measured by patch clamp recordings from similar neurons. The effect of exposure to different concentrations of Mp and some of its main ingredients was measured. RESULTS: Mp produced complex, dose dependent, reversible effects on network activity, increasing it with low concentrations, and decreasing it at high concentrations. Individual flavonoids contained in Mp mimicked the effects of the extract, both for the facilitating and suppressing effects of the extract. Electrophysiologically, Mp caused a reduction in spontaneous activity, but did not affect membrane properties of individual patch clamped neurons, nor did it affect mEPSCs recorded from these neurons. However, a transient increase in reactivity to pulse application of GABA was evident. CONCLUSIONS: These results suggest that a main sedative effect of Mp is on GABAergic neurotransmission in cultured hippocampal neurons.

Early exposure of cultured hippocampal neurons to excitatory amino acids protects from later excitotoxicity.

Status epilepticus occurring in early postnatal development protects CA1 hippocampal neurons, the region most sensitive to seizure-induced injury in the developing brain. Here, we developed a "two hit" model in dissociated cultures of the rat hippocampus to test whether pre-exposure of immature neurons to high concentrations of glutamate, N-methyl-D-aspartic acid (NMDA) or alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) during a relatively resistant period prevents neurons from dying following a second exposure to the same chemicals after neurons mature and become highly vulnerable to excitatory amino acids (EAAs). Cultures were exposed to varied doses of glutamate, NMDA, or AMPA for 48 h at 5 DIV and again at 14 DIV for 5, 15, or 30 min. NeuN immunohistochemistry showed early exposure to glutamate (500 microM) killed approximately half of the neurons (52+/-8.6%) compared to the marked depletion that occurs after one exposure at 14 DIV (98+/-0.79%). When cultures were first challenged with moderate doses of glutamate (200 microM) followed by the high dose 7 days later, a significant population of neurons was spared (35.3+/-1.2%). Similarly, pre-exposure to maximal doses of NMDA (100 microM) increased the proportion of surviving cells following the second challenge. In contrast, AMPA (100 microM) was equally toxic after early or late applications and did not protect from the second exposure. GluR1 subunit expression was markedly decreased at 48 h after one or two exposures to 200 microM glutamate (by 44.57+/-3.6%, 45.07+/-3.69%) whereas GluR2 subunit expression was reduced by a lesser amount (25.7 57+/-3.8%). Confocal microscopy showed that one or two exposures to NMDA caused GluR2 protein to downregulate even further whereas parvalbumin (PV) was dramatically increased in the same neurons by over four-fold. On the other hand, calbindin (CB) immunoreactivity was nearly absent after the first exposure to 500 microM glutamate. These data indicate that early, transient exposure to certain EAAs at high doses can induce long-lasting neuroprotection. Alterations in the GluR1/GluR2 ratio as well as differential expression of specific calcium binding proteins may contribute to this neuroprotection.