Effects of membrane potential on intracellular Cl⁻ accumulation.

<p><b>A.</b> Varying the rate of excitatory synaptic drive (<i>f</i>_exc) caused a depolarizing shift in <i>E</i>_Cl secondary to changes in average membrane potential. <i>f</i>_inh was fixed at 0.4 Hz. <b>B.</b> Spiking exacerbates intracellular Cl⁻ accumulation as illustrated here by convergence of the model to different steady state [Cl⁻]_i depending on whether the model does or does not contain HH channels (<i>i.e.</i> does or does not spike, respectively). For this simulation, KCC2 activity was low (10%), <i>f</i>_inh = 0.8 Hz, and <i>f</i>_exc = 0.4 Hz. <b>C.</b> Sample traces showing inter-relationship between [Cl⁻]_i and spiking. Neuron began spiking when constant excitatory current was applied to the soma, but without any concomitant change in [Cl⁻]_i since there was not yet any GABA_AR-mediated conductance. Turning on constant GABA_AR conductance in the soma terminated spiking, but at the expense of intracellular Cl⁻ accumulation. Chloride slowly accumulated over the next several seconds until membrane potential reached spike threshold, at which point spiking resumed and Cl⁻ began a second phase of accelerated accumulation. <b>D.</b> To test whether Cl⁻ accumulation is exacerbated by excitatory synaptic input in real neurons, somatic Cl⁻ concentration was measured using FLIM in neurons with or without glutamatergic receptor activation by kainate. As predicted by simulations, Cl⁻ accumulation was greater in neurons exposed to kainate. Furosemide was applied to block KCC2 activity in these experiments (**, p < 0.001; ***, p < 0.0001). Data from 56 cells from 5 coverslips. <b>E.</b> Comparison of input-output curve for static (<i>black</i>) vs. dynamic (<i>red</i>) <i>E</i>_Cl. Discrepancies between the curves clearly demonstrate that <i>E</i>_Cl cannot be approximated as constant value when considering a range of input conditions.