SAT-427 Low Chloride Transporter Expression in Vasopressin Neurons

Several membrane proteins function as ion pumps/transporters that work to maintain the relative ionic concentrations necessary for neuronal signaling. The potassium-chloride co-transporter two (KCC2) is a symporter that actively pumps K(+) and Cl(-) out of the cell in order to maintain a low intracellular [Cl(-)], while the sodium-potassium-chloride cotransporters one and two (NKCC1/NKCC2) are symporters that actively pump Na(+), K(+), and Cl(-) into the cell. KCC2 and NKCC1/2, therefore, have opposing actions on Cl(-) transport. In the adult, high KCC2 expression leads to low intracellular [Cl(-)], which causes GABA(A) receptor Cl- channels to flux Cl(-) into the neuron, causing a membrane hyperpolarization. Early in development however, low KCC2 expression reverses the Cl(-) gradient and causes GABA(A) receptors to flux Cl(-) out of the neuron, leading to membrane depolarization and neuronal excitation. The change in the polarity of GABA signaling during development is, in part, due to a shift in KCC2 expression from low to high with the maturation of synaptic circuits. Our previous studies have demonstrated an excitatory GABA(A) receptor-mediated response due to a GABA equilibrium potential (E(GABA)) that is shifted positive, like that seen early in development, in adult vasopressin (VP)-secreting neurons of the rat hypothalamus. In the current study, we used immunohistochemistry to compare expression levels of the main C(l-) transporters, KCC2, NKCC1, and NKCC2, between VP- and oxytocin (OT)-secreting neurons of the hypothalamic paraventricular and supraoptic nuclei. We found that, in adult male Wistar rats, expression of all three transporters is uniformly lower in VP neurons than in adjacent OT neurons in the hypothalamus. Because the phosphorylation state of KCC2 affects membrane trafficking and stabilization, we are also using Western blot to analyze phosphorylation of KCC2 under different osmotic conditions. Finally, we are using Cre-dependent viral transduction to express a light-activatable Cl(-) channel conditionally in vasopressin-secreting neurons to test for the effect of Cl(-) flux on vasopressin secretion.