Distinct properties of functional KCC2 expression in immature mouse hippocampal neurons in culture and in acute slices

Abstract

A hallmark in the development of GABAergic neurotransmission is the switch in GABA(A)-mediated responses from depolarizing to hyperpolarizing. This occurs due to a gradual decrease in the intracellular concentration of chloride caused by the functional expression of the neuron-specific K-Cl cotransporter KCC2. Whether a mere increase in the amount of KCC2 protein is the rate-limiting step in vivo, or a further activation of the otherwise nonfunctional cotransporter is required, is not clear. Imposing a fixed Cl(-) load via patch pipette we measured the resultant somato-dendritic gradients in reversal potential of GABAergic currents to determine the time course of functional maturation of KCC2-mediated Cl(-) extrusion in two preparations: cultured mouse hippocampal neurons plated at embryonic day 17 and CA1 pyramidal cells in acute slices. We found that in immature neurons in both preparations the gradient is initially small or not detectable. It undergoes an abrupt increase at around days 13-14 in culture, while a more gradual increase occurs between postnatal days 5-14 in slices. Consistent with the presence of a nonfunctional form of KCC2 in immature hippocampal neurons grown in culture, application of the broad-spectrum kinase inhibitor staurosporine produces a rapid and potent up-regulation of KCC2 function in these cultured neurons, but not in neonatal slices. Taken together with our previously published data, these results indicate that the functional activity of KCC2 in vivo parallels the developmental expression of the protein, whereas cultured neurons require an additional activation step (mimicked by staurosporine) for KCC2 to become functional.

Simultaneous two-photon imaging of intracellular chloride concentration and pH in mouse pyramidal neurons in vivo

“…5D). These data are in agreement with measurements of the ion transport efficacy of KCC2 in cortical neurons in vitro, which show strong up-regulation commencing at around P6, with near-maximum extrusion rates achieved at ∼P14-P16 (3,36). The high [Cl − ] i in immature neurons is generally thought to be maintained by Cl − uptake via NKCC1 (in the Introduction).…”

Neto2 is a KCC2 interacting protein required for neuronal Cl − regulation in hippocampal neurons

“…5), so we next asked whether this residual protein was still capable of extruding Cl − . Functional changes in the Cl − extrusion capacity of KCC2 can be examined in the presence of an ionic load (8,26,27). We examined the function of KCC2 in Neto2-null mice by loading the neuronal soma with 50 mM Cl − (using a patch pipette).…”

Activity-Dependent Cleavage of the K-Cl Cotransporter KCC2 Mediated by Calcium-Activated Protease Calpain

“…Indeed, control measurements of somatic E GABA yielded a value of Ϫ40.7 Ϯ 0.41 mV (n ϭ 26), demonstrating that the somatic [Cl Ϫ ] i of CA1 pyramidal neurons was clamped at the [Cl Ϫ ] of the pipette. Thus, under these conditions, a negative deviation of the dendritic E GABA from the somatic value (i.e., ⌬E GABA ) is a quantitative measure of the efficacy of Cl Ϫ extrusion (Jarolimek et al, 1999;Khirug et al, 2005). Somatic and dendritic E GABA values were determined from the current-voltage rela-tions, obtained by sequentially clamping the membrane potential at different holding potentials with a 5 mV increment and a 10 s step interval.…”

“…Somatic and dendritic E GABA values were determined from the current-voltage rela-tions, obtained by sequentially clamping the membrane potential at different holding potentials with a 5 mV increment and a 10 s step interval. Caged GABA was photolyzed 50 ms after the start of each voltage step to evoke local GABA A receptor-mediated currents (Khirug et al, 2005). To this end, 1 mM DPNI-caged GABA dissolved in extracellular solution was delivered at a flow rate of 1-2 l/min via an UltraMicroPump II (WPI) equipped with a syringe and a 100 m inner tip diameter quartz needle (WPI) and locally photolyzed using the 375 nm output of a continuous emission diode laser (Excelsior 375; Spectra Physics).…”

“…To study the effect of blocking protein synthesis on the functional KCC2 pool, we used an electrophysiological assay to measure KCC2-mediated Cl Ϫ extrusion (Khirug et al, 2005;Li et al, 2007). Because of the similar results obtained with the two protein synthesis inhibitors in the biochemical assays (Fig.…”