Illuminating the Role for Chloride Dysregulation in Network Activity

The Contribution of Raised Intraneuronal Chloride to Epileptic Network Activity.

Alfonsa H, Merricks EM, Codadu NK, Cunningham MO, Deisseroth K, Racca C, Trevelyan AJ. J Neurosci 2015;35:7715–7726.

Altered inhibitory function is an important facet of epileptic pathology. A key concept is that GABAergic activity can become excitatory if intraneuronal chloride rises. However, it has proved difficult to separate the role of raised chloride from other contributory factors in complex network phenomena, such as epileptic pathology. Therefore, we asked what patterns of activity are associated with chloride dysregulation by making novel use of Halorhodopsin to load clusters of mouse pyramidal cells artificially with Cl⁻. Brief (1–10 s) activation of Halorhodopsin caused substantial positive shifts in the GABAergic reversal potential that were proportional to the charge transfer during the illumination and in adult neocortical pyramidal neurons decayed with a time constant of τ = 8.0 ±2.8s. At the network level, these positive shifts in EGABA produced a transient rise in network excitability, with many distinctive features of epileptic foci, including high-frequency oscillations with evidence of out-of-phase firing (Ibarz et al., 2010). We show how such firing patterns can arise from quite small shifts in the mean intracellular Cl⁻ level, within heterogeneous neuronal populations. Notably, however, chloride loading by itself did not trigger full ictal events, even with additional electrical stimulation to the underlying white matter. In contrast, when performed in combination with low, subepileptic levels of 4-aminopyridine, Halorhodopsin activation rapidly induced full ictal activity. These results suggest that chloride loading has at most an adjunctive role in ictogenesis. Our simulations also show how chloride loading can affect the jitter of action potential timing associated with imminent recruitment to an ictal event (Netoff and Schiff, 2002).

Commentary

Perturbation in GABAergic inhibition is often a prominent feature of epilepsies, and treatments aimed at enhancing GABA currents are part of the standard clinical arsenal to stop seizures. Apart from loss of interneurons, functional deficits in inhibition are also known to occur in epilepsy (1). GABA-mediated inhibition in the mammalian brain largely depends on the Chloride ion (Cl⁻) gradient (2). Gradient driven influx of negatively charged Cl⁻ into the cell hyperpolarizes the cell membrane, thereby driving the membrane away from the threshold for action potential generation. Physiologically, homeostasis of Cl⁻ is maintained by cation-chloride cotransporters (CCC) NKCC1 and KCC2, which regulate Cl⁻ influx and efflux. Dysregulation of CCCs leads to intracellular accumulation of Cl⁻ and a positive shift in GABA reversal potential (E_GABA). Interestingly, dysregulation of NKCC1 and KCC2 have been observed in human temporal lobe epilepsy and in animal models (3–6). However, whether such a reversal in the polarity of GABA alone would be sufficient to initiate seizures is still not clear. The study by Alfonsa and coworkers is aimed at answering this question.

Alfonsa and coworkers make novel use of optogenetic technology, which involves expression of light-activated channels or transporters to control neuronal activity, to manipulate Cl⁻ levels in cortical pyramidal neurons. Activation of halorhodopsin (NpHR) by amber light induces membrane hyperpolarization by transporting Cl⁻ into the cell. The intracellular chloride load, deemed an unfortunate side effect due to its potential impact on GABA currents, spearheaded the development of Archaerhodopsin (Arch), which extrudes protons on light activation without altering chloride homeostasis (7). The authors have brilliantly used halorhodopsin and Arch to address the role of intracellular Cl⁻ dysregulation in seizure induction. They effectively distinguish between effects of hyperpolarization, which is common to both opsins, from those due to the Cl⁻ dysregulation, which is unique to halorhodopsin. Using this strategy together with electrical and chemical activation of slices and computational modeling, the authors examine how enhancing intracellular Cl⁻ impacts network rhythms and seizure generation.

Using perforated patch recordings, which prevent exchange of chloride from recording solution to the cell, the authors first determine the magnitude and duration of shift in E_GABA following light activation in cultured cortical neurons transfected with NpHR. By evaluating E_GABA from responses to voltage ramps applied during agonist-evoked GABA transients before and after NpHR activation, they demonstrate a consistent depolarizing shift in E_GABA after NpHR activation, which slowly recovers to baseline over 10 seconds. They confirm that pyramidal neurons in cortical slices from mice transfected with NpHR demonstrate a depolarizing shift in E_GABA for over 10 seconds after light activation while neuronal excitability returns to baseline levels within a second. Thus, they identify a brief duration following NpHR activation when the membrane properties are normal and E_GABA remains depolarized. In parallel, they show that light activation leads to hyperpolarization without altering E_GABA in Arch-transfected neurons. These findings validate the novel use of NpHR to perturb E_GABA, characterize the temporal window of E_GABA dysregulation, and justify using Arch in control studies.

Next, in cortical slices perfused with low-divalent cations, to promote network activity, they demonstrate that NpHR priming by repeated light activation consistently enhances stimulus-evoked network activity after the end of light activation. Specifically, NpHR and not Arch priming resulted in emergence of high frequency (75–600 Hz) oscillations with enhanced power in the 300 to 600 Hz range and increased multiunit firing, indicating a role for Cl⁻ loading in these effects. The authors focused on the 75 to 300 Hz frequency band to examine the possible role for inhibition in generating this abnormal network activity following NpHR priming. By evaluating the phase of the multiunit activity with respect to the subthreshold oscillation, they show that NpHR, but not Arch priming, increases temporal jitter in neuronal firing at the oscillation peak, and in a subset of slices, promotes out-of-phase firing in a cohort of neurons. In carefully constructed analytical controls, they show that the out-of-phase firing was not a result of spectral leak indicating that the effect was a result of chloride dysregulation. In a simple multi-compartmental spiking neuron model with noisy dendritic glutamatergic inputs and high frequency somatic GABAergic inputs, they demonstrate that systematically changing E_GABA from −75 to −45 mV progressively increases spike time jitter eventually resulting in out-of-phase firing when E_GABA crosses action potential threshold. Next they used model neuron firing, simulated to include synaptic inhibition at different E_GABA levels, to predict the phase distributions of firing in cell populations. When model neuron firing was drawn from simulations with normally distributed E_GABA, in a range subthreshold for generation of action potentials, the predicted firing of cell populations was in-phase. However, imposing a skewed E_GABA distribution including cells with E_GABA positive to firing threshold, as noted in human epileptic neurons (4), resulted in out-of-phase firing in a subset of neurons (8, 9). Although high-frequency oscillations in NpHR-primed slices appeared similar to those in epilepsy, NpHR priming failed to initiate seizures following low-frequency electrical stimulation in a low-cation medium demonstrating that a depolarized E_GABA is not sufficient to initiate seizures. However, NpHR-priming was found to facilitate seizure induction by 4-aminopyridine demonstrating that chloride loading augments seizure generation in susceptible networks.

The innovative use of halorhodopsin and the careful validation of this methodology will enable future use of this technique to examine cell specific chloride dysregulation. While the authors compare the E_GABA variability in NpHR-primed cultured neurons with that recorded in epileptic tissue (4), variability in the current study resulted from changing the duration of NpHR activation, and slice experiments with 2-second light exposure showed limited E_GABA variability. Whether a constant duration of NpHR priming in slice experiments could elicit the E_GABA variability needed to support out-of-phase activity was not examined. It is possible that even with constant priming, differences in NpHR expression among transfected neurons was sufficient to drive E_GABA above firing threshold in a subset of neurons and support out-of-phase activity. It is worth noting that chloride dysregulation in fast-spiking interneurons can impact oscillations (10), and viral transfection can cause nonspecific expression of NpHR in GABAergic neurons. Future studies should examine the mechanisms underlying the 300 to 600 Hz oscillation induced by NpHR priming and determine if NpHR priming could initiate seizures in pathologically altered networks in experimental epilepsy.

In summary, the study introduces an elegant method to perturb cellular chloride and examine the effects of altered GABA reversal potential on network activity. The authors demonstrate that while chloride loading and resulting depolarizing GABA might enhance precipitation of seizures, they are not sufficient to initiate seizures. In addition, these results demonstrate that depolarized E_GABA, which crosses firing threshold in a subset of neurons, could contribute to high frequency oscillations in epileptic circuits. Overall, the study sheds light on a long standing open question and emphasizes the role for multiple converging mechanisms in seizure initiation.