Regulated Expression of Rat Hippocampal Chloride Transporters During the Early Postnatal Period

Matthew Marcetich
Marquette University
Mentor: Dr. Michelle Mynlieff

In multiple studies, the function of the neurotransmitter GABA has been shown to switch from excitatory during early development to inhibitory into adulthood.  This functional switch is likely due to the presence of a high intracellular chloride concentration created by high expression of NKCC1 (chloride importer) and low expression of KCC2 (chloride extruder) in immature neurons, and a low intracellular chloride concentration created by low expression of NKCC1 (chloride importer) and high expression of KCC2 (chloride extruder) in mature neurons.  Additionally, influx of calcium through L-type calcium channels has been associated with the change in the chloride reversal potential observed between birth and adulthood in Sprague-Dawley rats, and is facilitated by GABAB activation.  Thus, our goal is to examine how GABAB activation and L-type calcium current affect the expression of NKCC1 and KCC2 during the early postnatal period of Sprague-Dawley rats.  Western blot analysis using PVDF membrane was conducted using hippocampal protein extractions from both whole tissue (from rats aging 1 day to 6 weeks) and cultured rat hippocampal cells (isolated on postnatal day 0 and maintained for 1-15 days).  Polyclonal and monoclonal anti-NKCC1 antibodies and polyclonal anti-KCC2 antibodies were used for identifying NKCC1 and KCC2 within the protein preparations.  Protein content in each preparation was determined using a BCA protein assay, and proteins were separated using SDS-PAGE with 3-8% Tris-Acetate gels.  We have found that NKCC1 expression is relatively high during the first postnatal week (peaking around day 8), and decreases sharply during the second postnatal week to low steady-state levels of expression into adulthood, in vivo.  Conversely, we have found that KCC2 expression increases during the first two postnatal weeks (peaking around day 12) and high steady-state levels are sustained into adulthood, in vivo. Future studies will examine NKCC1 and KCC2 expression within cultured hippocampal cells treated with baclofen (GABAB agonist), saclofen (GABAB antagonist) and nimodipine (L-type calcium channel antagonist) for one week using cells isolated on postnatal day 0.
 
 
 

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