Background γ-Aminobutyric acid (GABA) is well known for its inhibitory effects in the mature central nervous system (CNS). However, in developing neonatal neurons, GABA-mediated processes are excitatory rather than inhibitory. This excitatory GABAergic transmission in the neonate has been linked to critical steps in neuronal development. At a critical point in neuronal maturation, GABAergic transmission reverses from excitatory in the developing brain to inhibitory in the more mature brain. This developmental switch in GABA-regulated transmission is coupled to an induction in the expression of the neuronal chloride ion (Cl-) extruding K+/Cl− cotransporter KCC2 perinatally. As the CNS matures, expression of KCC2 increases, causing a shift in resting Cl− concentrations from highly concentrated intracellularly in immature brains to high extracellular concentrations in mature brains. Alcohol has long been known for its potentiating effects on GABAergic transmission. Furthermore, potentiation of GABAergic transmission has been linked to an increase in expression of KCC2. Thus, KCC2 is a potential target for defects seen in alcohol-exposed developing neurons. The objective was to examine the effects of alcohol exposure on developing neurons and expression of KCC2 as a possible mechanism for sequelae of fetal alcohol exposure.
Methods Coronal brain slices from neonatal Sprague-Dawley rats were collected in consecutive days (P3, P4, P5, and P6). Right and left sister slices were randomly divided into the ethanol or control group. The EtOH slices were incubated in 50 mM EtOH + oxygenated ACSF for 4 hours; the control group was incubated in oxygenated ACSF only. Hippocampi were isolated and homogenized and a crude membrane fraction prepared by centrifugation. Western immunoblot assays were run with rabbit polyclonal anti-KCC2 antibodies. KCC2 optical density was normalized against Coomassie-stained bands.
Results In vitro EtOH exposure significantly decreased KCC2 protein levels in membrane preparations of microdissected hippocampi compared with control.
Conclusion Despite the initial hypothesis that EtOH, given its GABA-potentiating effects, would increase the expression of KCC2 altering the critical timetable in neuronal development, the results indicated the contrary. This finding appears to contradict previous data demonstrating that enhanced potentiation of GABAergic activity elevates KCC2 expression in immature neurons. Therefore, EtOH might be regulating the expression of KCC2 via other mechanisms, possibly via mechanisms similar to those induced after excessive excitatory activity or neuronal cell injury in the adult CNS.
Statistics from Altmetric.com
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.