Elsevier

Neuroscience

Volume 340, 6 January 2017, Pages 530-541
Neuroscience

Long-term alcohol exposure elicits hippocampal nonsynaptic epileptiform activity changes associated with expression and functional changes in NKCC1, KCC2 co-transporters and Na+/K+-ATPase

https://doi.org/10.1016/j.neuroscience.2016.11.015Get rights and content

Highlights

  • Nonsynaptic effects of long-term alcohol exposure (3 months) on the hippocampus were investigated.

  • Alcohol exposure changed NKCC1, KCC2 and Na-K-ATPase expressions.

  • NEA enhanced due to alcohol exposure.

  • Increase in DC shift is due to NKCC1 action.

  • Neurodegeneration observed in the treated groups diminished with NKCC1 blockage.

Abstract

Nonsynaptic mechanism changes, particularly the enhancement of NKCC1 expression in the dentate gyrus (DG) after 4 weeks of ethanol consumption, motivate the present work, in which rats were submitted to a period of chronic consumption (12 weeks). Four groups of six animals (6-week-old male Wistar rats) were formed, including the control (C), ethanol 1 (E1), ethanol 2 (E2) and ethanol 3 (E3) groups. The rats in the E1, E2 and E3 groups were treated daily with a 30% v/v solution of ethanol, administered via oral gavage (1.0, 2.0 and 3.0 g/kg, respectively). Nonsynaptic epileptiform activities (NEA) were induced by means of the zero-Ca2+ and high-K+ model using hippocampal slices and were recorded in the DG. The presence of NKCC1, KCC2, α1-Na+/K+-ATPase and GFAP immunoreactivity was analyzed. The results demonstrate that alcohol consumption changes NEA, and these changes are more prominent at the lower dosage. An increase in the DC shifts associated with epileptiform discharges was present with the low dose. This increase was correlated with the increment of NKCC1 expression. Confocal microscopy images indicate the NKCC1 increase was pronounced in the initial axonal segment of granule cells. The blockage of these cotransporters during NEA induction with bumetanide suppressed the DC shift increase and diminished all parameters of NEA that were quantified for all groups treated with ethanol. Therefore, the increase in NKCC1 expression and the effective activity of this cotransporter, which were observed in the treated groups, suggest that drugs that act for block NKCC1 represent promising strategies for diminishing the effects of alcohol damage on the brain.

Introduction

Several clinical reports provide evidence of convulsive seizures in alcoholics (Rehm et al., 2010, Zuccoli et al., 2010). Following chronic ethanol consumption, compensatory mechanisms may be responsible for the appearance of morphological and physiological changes. With continuous alcohol-use, inflammatory processes may enhance and change the ionic homeostasis of the neuronal tissue, thereby increasing susceptibility to seizures and neurodegeneration (Collins et al., 1998, Santos et al., 2013).

Alcohol-use investigations and its effects on the limbic system indicate neurodegeneration processes and the corresponding commitments of the cognition and memory processes (Nixon, 2006). Recent studies have focused attention on nonsynaptic mechanisms of ethanol in the generation of seizures, including its relationship with neuronal and glial functions as well as its performance in the transmembrane ionic equilibria (Collins and Neafsey, 2012, Tajuddin et al., 2013, Santos et al., 2013).

Acting as a solvent of cell membrane phospholipids, ethanol promotes the destabilization of immersed proteins and subsequent alteration of ion transport mechanisms. Ethanol exposure causes cell water elevation, brain swelling and neuroinflammation (Collins et al., 1998, Ingólfsson and Andersen, 2011). Studies have demonstrated that the manipulation of the extracellular space volume in brain slice preparations via osmotic challenge induces an increased volume of neural cells and have demonstrated the critical role of the extracellular space fraction in the processes of excitotoxicity (Zador et al., 2009). Observations also indicate a close link between alcoholism and status epilepticus, which is mediated by a nonsynaptic mechanism and accumulation of electrolytes (Collins et al., 1998, Santos et al., 2013). Cellular edema and neuronal hyper-synchronism may be prevented by furosemide, with or without the presence of Ca2+ (Hochman et al., 1995, Collins et al., 1998). This may be an indication that nonsynaptic mechanisms are involved and may include the cation-chloride cotransporters KCC2 and NKCC1, which are both blocked by furosemide (Margineanu and Klitgaard, 2006, Blaesse et al., 2009). Alterations in Cl homeostasis as a result of an unbalance between KCC2 and NKCC1 have been reported (Ben-Ari, 2002, Kahle et al., 2008). Recent evidence indicates that morphological changes in the expression of cation-chloride cotransporters are modulated by ethanol exposure and may have a direct effect on the susceptibility to induce epileptiform discharges (Santos et al., 2013).

The zero-calcium model of epileptiform activity is an in vitro model of ictal-like discharges (Jefferys and Haas, 1982, Taylor and Dudek, 1982). The activity induced with this model closely mimics in vivo recordings, with events that last a few seconds to tens of seconds. In the absence of calcium, these activities are not based on the synaptic circuitry because the chemical synapses are blocked. The induction of nonsynaptic epileptiform activity in the dentate gyrus (DG) demands increased extracellular [K+] compared with the CA, which supports the hypothesis of the DG functioning as a closed “gate”, which prevents seizure activity from entering the CA (Walther et al., 1986, Dreier and Heinemann, 1991, Lothman et al., 1992). However, the DG may lose its protective role during a phenomenon referred to as “maximum dentate activation” (MDA), which is modeled by a zero-calcium and high potassium condition, when the region becomes an amplifier of seizure activity (Lothman, 1991, Lothman et al., 1991, Lothman et al., 1992).

Santos et al. (2013) evaluated the effect of 4 weeks of ethanol consumption on ictogenesis in the rat brain. Using the zero-calcium model, the authors identified an enhancement of ictal-like discharges induced in the DG of hippocampus slices. Increased NKCC1 expression, cell loss, edema and gliosis were associated with these changes. With the aim to investigate the progression of these changes during a longer period of consumption and their reflex on ictogenesis and neurodegeneration, in the present work we investigated the effect of ethanol consumption for 12 weeks. The study assessed the nonsynaptic epileptiform activities (NEA) induced in the DG of hippocampus slices. Using confocal laser microscopy, the expression of cotransporters and Na+/K+-ATPase was also investigated. The increment of the cotransporter expression was determined, and the effects of NKCC1 increasing the ictogenesis of the group treated with a low dosage of ethanol and the neurodegenerative process after ictal-like activity on the group treated with a high dosage were demonstrated. The blockage of NKCC1 with bumetanide resulted in a reduction in neurodegenerative parameters, such as nucleus pyknotic and whitish zones in the granule layer.

Section snippets

Animals

The institutional animal care and use committee (CEUA of the Federal University of São João del-Rei) approved the protocol (3/2011) for the present study. Water and lab chow were provided ad libitum, and the room temperature (20–22 °C), light (12 h), and humidity (50–55%) were controlled.

Four groups with seven animals (6-week-old male Wistar rats) were formed, including the control (C), ethanol 1 (E1), ethanol 2 (E2) and ethanol 3 (E3) groups. The rats in the E1, E2 and E3 groups were treated

Effect of GABAa receptor blockage on nonsynaptic epileptiform activity

To investigate whether the GABAa receptor would modulate cotransporter activity, PTX (100 μM) was used to block this receptor during nonsynaptic epileptiform activity induction (n = 10 animals). As shown in Fig. 1, no significant changes were identified in the quantified electrographic parameters after the blockage (DC shift: 10.72 ± 1.06 mV, before; 11.05 ± 1.19 mV, after PTX, t = 1.239; df = 9; p = 0.2466; ED: 40.01 ± 4.57 s, before; 38.93 ± 5.18 s, after PTX; t = 0.6589; df = 9; p = 0.5264; PS: 14.22 ± 1.17 mV before,

Discussion

The DG is resistant to epileptiform activity in in vitro convulsant models (Schwartzkroin and Prince, 1978, Fricke and Prince, 1984, Mody et al., 1987). However, under favorable conditions, this seizure-resistant area may be transformed to an area able to sustain robust epileptiform activity that resembles the MDA (Lothman, 1991, Lothman et al., 1991, Lothman et al., 1992).

In the present investigation, despite changes in the physical conditions of the alcohol-treated animals were observed, no

Conclusion

The present findings indicate the complex interplay between NKCC1 and KCC2 modulation of the nonsynaptic mechanism alterations induced by the long-term consumption of alcohol. Consumption at a lower dosage reflects nonsynaptic changes that enhance the conditions that sustain ictogenesis. However, consumption at higher dosages is associated with an intense neurodegenerative state that may reflect reduced neuronal functionality and therefore decreased ictogenesis. The main conclusion of the

Acknowledgments

Support to conduct this work was provided by Fapemig (Fundação de Amparo à Pesquisa do Estado de Minas Gerais), CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico), FAPESP (Fundação de Amparo à Pesquisa do Estado de São Paulo) and CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior).

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