Neurotoxicology. 2015 Mar;47:62-71. doi: 10.1016/j.neuro.2015.01.005.

Chronic exposure to alcohol alters network activity and morphology of cultured hippocampal neurons.

Korkotian E1, Botalova A2, Odegova T2, Segal M1.

1 Department of Neurobiology, Weizmann Institute of Science, Rehovot, Israel

2 Department of Immunology, Perm state University, Perm, Russia

 

ABSTRACT

The effects of chronic exposure to moderate concentrations of ethanol were studied in cultured hippocampal neurons. Network activity, assessed by imaging of [Ca(2+)]i variations, was markedly suppressed following 5 days of exposure to 0.25-1% ethanol. The reduced activity was sustained following extensive washout of ethanol, but the activity recovered by blockade of inhibition with bicuculline. This reduction of network activity was associated with a reduction in rates of mEPSCs, but not in a change in inhibitory synaptic activity. Chronic exposure to ethanol caused a significant reduction in the density of mature dendritic spines, without an effect on dendritic length or arborization. These results indicate that chronic exposure to ethanol causes a reduction in excitatory network drive in hippocampal neurons adding another dimension to the chronic effects of alcohol abuse.

KEYWORDS: Calcium; Dendritic spines; Ethanol; Hippocampus culture; Synapses

PMID: 25655208 [PubMed – indexed for MEDLINE]

 

Supplementary 

Alcoholism constitutes a major burden in modern society. The chronic effects of alcohol consumption involve cognitive, motor and emotional deterioration, associated with neurodegeneration in critical brain regions. Animal models do not provide a sufficient understanding of the lasting molecular and cellular effects of chronic exposure to alcohol. We have developed an in-vitro model, consisting of cultured primary hippocampal neurons, maintained in-vitro for several weeks. These cells are spontaneously active, and generate ongoing synchronous network bursts, that can be easily measured using calcium indicators. The neurons can be exposed to known concentrations of ethanol for various durations, from minutes to days, and the effects of ethanol, as well as its removal, can be measured. In the first series of studies we exposed the cells to low (0.25-0.5%) ethanol concentrations to find an increase in synchronized network bursts, and a decrease in the duration of individual bursts. Higher concentrations of ethanol eliminated network activity. These effects were reversible upon wash. The effects of the high, but not the low ethanol were blocked by the GABA antagonist bicuculline, indicating an activation of the GABAergic receptor. The enhancing action of low ethanol was blocked by apamin, an SK potassium channel antagonist, and mimicked by 1-EBIO, an SK channel opener. It was proposed that in cultured hippocampal networks low concentration of ethanol is associated with SK channel activity, rather than the GABAergic receptor.

 

 

EK fig1Figure 1: Schematic illustration of the proposed loci of action of ethanolat the synapse. Presynaptic terminal releases glutamate which activates NMDA and AMPA receptors postsynaptically. Intracellular calcium concentration is enhanced by activation of voltage gated calcium channels (VGCC), NMDA receptors and release of calcium from endoplasmic reticulum stores. The increase in intracellular calcium activates SK channels. Ethanol is proposed to act by enhancing activity of SK channels, an effect that is blocked by apamin. Not shown here is the involvement of GABAergic receptors, which are facilitated by higher concentration of ethanol.

 

We then proceeded to study the effects of chronic exposure to physiological concentrations of ethanol in hippocampal culture. It was found that neuronal activity could be significantly increased following 5-24 hours of exposure to 0.25-0.5% ethanol while almost completely blocked following 5 days of exposure. Interestingly, both effects have persisted even after extensive wash. In its turn, reduced activity was found to be sensitive to GABAergic receptor antagonist bicuculline related to the blockade of inhibition.

Additionally the morphology of dendritic spines was tested in control conditions and in the presence of ethanol. A significant reduction in the density of mature mushroom dendritic spines could be found in hippocampal cultures treated with alcohol during 5 days.

Our results indicate that chronic exposure to ethanol causes dual homeostatic effect on the network drive in hippocampal neurons. Its direction strictly depends on the duration of chronic exposure while it persists even after the direct influence of the substance is over. The hippocampal network in the dish thus provides a useful test system for the examination of the effects of chronic alcohol and their amelioration by selective drugs of interest.

 

EK fig2

Figure 2: Schematic illustration of the membrane receptors proposed to be affected by ethanol, the SK channel, and the GABA-linked chloride channel.

 

References:

Korkotian E, T Bombela, T Odegova, P Zubov, and M. Segal. Ethanol affects network activity in cultured rat hippocampus: mediation by potassium channels. PLoS One Nov 2013 e75988.

Korkotian E, Botalova A, Odegova T, Segal M Chronic exposure to alcohol alters network activity and morphology of cultured hippocampal neurons. Neurotoxicology. 2015 Feb 2;47C:62-71. doi: 10.1016/j.neuro.2015.01.005.

 

Contact:

Eduard Korkotian, Ph.D.

Dept. of Neurobiology, The Weizmann Institute, Israel

eduardkorkotian@gmail.com

 

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