Data are based on simulations in a model of a spinal lamina I neuron (see Prescott et al., 2006 for details). (A) Output firing rate (f_out) is plotted against the rate of excitatory synaptic input (f_exc). Gray line shows input-output curve for no inhibition. Black lines show input-output curve for inhibition (whose input frequency is proportional to f_exc) for different values of E_anion. “Inhibitory” input reduces spiking when E_anion = −65 mV, but fails to reduce spiking when E_anion = −55 mV, and actually increases spiking when E_anion = −50 mV although spike threshold is −49 mV. (B) Despite its effect on firing rate, inhibition reduced average depolarization even for E_anion = −50 mV. Thus, there is a discrepancy between modulation of firing rate and modulation of membrane potential, which is explained in C. (C) For equivalent depolarization, the model with inhibition spiked faster than the model without inhibition, regardless of the value of E_anion. (D) The effect in C is explained by shortening of the membrane time constant (τ_m) that accompanies the increase in membrane conductance caused by inhibitory input. (E) The shunting-induced change in passive membrane properties influence how the neuron responds to inputs with different frequencies, as shown here by power spectral analysis of the voltage response to synaptic input. The important point is summarized in the inset, which shows that the inhibited neuron (with shorter τ_m, black line) charges its membrane faster, and is therefore able to spike faster, than the neuron without inhibition (gray line). Thus, inhibition can ironically increase spiking even while reducing depolarization. Modified from Prescott et al. (2006).

Signaling events that may increase NKCC-mediated Cl⁻ influx include phosphorylation (star 1), protein trafficking (star 2) or decreases in intracellular Na⁺ (star 3). Star 1: In contrast to KCC2, phosphorylation of NKCC1 results in an increase in activity, CaMKIIα (downstream of mGluR1/5), PKCδ and ERK have all been shown to increase NKCC1 activity, although it is not known if these kinases directly phosphorylate NKCC1. JNK also increases NKCC1 activity via direct phosphorylation of the co-transporter. A separate family of kinases, which are regulated by tonicity and cellular stress, positively modulate NKCC1. WNK1/4 act via SPAK and/or OSR1 to phosphorylate NKCC1. WNK3 also increases Cl⁻ accumulation via NKCC1 and decreases KCC2 activity, which augments the net effect of NKCC1 stimulation in neurons that express both co-transporters. It is not known if WNK3 directly phosphorylates NKCC1 (and KCC2) or if it acts through SPAK/OSR1. SPAK and OSR1 interact with the N-terminus of NKCC1, where the co-transporter is phosphorylated, making these kinases ideal candidates as integrators of signaling that converges on NKCC1. Star 2: Dynamic regulation of the density of NKCC in the cell membrane may also influence rates of Cl⁻ influx. An increase in intracellular cAMP concentration increases membrane delivery of NKCC2 in a Vamp2/3-dependent fashion. It is not known if this mechanism is also active at NKCC1. Star 3: A drop in the concentration of intracellular Na⁺ secondary to an increase in Na⁺/K⁺-ATPase may result in an increase in NKCC1 activity. This sequence of events may follow the increase in Na⁺ that occurs in association with an intense burst of neuronal activity. Star A, B and C: Persistent nociceptor activation caused by an intracolonic irritant injection causes an increase in dorsal horn NKCC1 phosphorylation (Star A) and an increase in NKCC1 in the cell membrane (Star B). It is not known if nociceptor spiking associated with this stimulus also contributes to an increase in NKCC1 activity in the dorsal horn (Star C).

There are at least 3 models that could explain the apparent paradox associated with the continued analgesic efficacy of benzodiazepine receptor agonist in the face of a shift in the valence of GABA_A receptor antagonist effects in the presence of persistent inflammation. All 3 require a functional separation between depolarizing shifts in Cl⁻ equilibrium potential and γ2 containing GABA_A receptors (i.e., those receptors responsive to benzodiazepines). Panel A: Separation in two subpopulations of dorsal horn neurons: In this model, γ2 containing GABA_A receptors are enriched in a subpopulation of dorsal horn neurons critical for the establishment of nociceptive threshold and/or the perception of pain (i.e., dorsal horn projection neuron, star 1). There is no shift in E_anion in these neurons, so that GABA_A receptor activation is inhibitory, and facilitation of this receptor activation by benzodiazepines is analgesic (star 1). The γ2-containing neuron is post-synaptic to both GABAergic input and excitatory interneurons. A depolarizing shift in E_anion in these excitatory interneurons results in a shift in the valence of GABA_A signaling and a GABA-mediated facilitation of nociceptive behavior (star 2). The density of γ2-containing GABA_A receptors in these interneurons would have to be relatively low such that benzodiazepines do not facilitate excitatory GABAergic transmission. The majority of lamina I–II neurons are interneurons (~90%) and these neurons may account for reduced KCC2 expression and excitatory synaptic GABA_A responses (star 2) observed following peripheral nerve injury (PNI). Such a model could also account for changes observed in the presence of inflammation if the GABA_A receptors on these excitatory interneurons have a higher affinity for muscimol. Panel B: Separation within a dorsal horn neuron: A differential distribution of γ2 containing GABA_A receptors and cation-Cl⁻ co-transporters within a dorsal horn neuron critical for nociceptive behavior and the perception of pain could underlie relatively localized changes in E_anion. In this model, there would be little or no change in E_anion around γ2 containing receptors following tissue injury (star 1), whereas a depolarizing shift in E_anion near receptors with a high affinity for muscimol would confer the facilitation of nociceptive transmission observed in the presence of persistent inflammation (star 2). Importantly, such a model is unlikely to account for changes observed following PNI given that a depolarizing shift in E_anion appears to impact the response to both exogenous and endogenous GABA_A receptor activation, suggesting that the shift in E_anion is relatively uniform in these neurons. Panel C: Separation with the central terminals of primary afferent neurons. This model is analogous to that described for Panel B, except that the separation of γ2-containing receptors and depolarizing shifts in E_anion occur in the primary afferent. A subset of dorsal horn interneurons make axo-axonic synapses onto incoming afferent fibers (star 1). While the subcellular localization of GABA_A subunits and/or NKCC1 is not known in these afferent axons, mechanisms involving the localization of midazolam-sensitive (γ2-containing) GABA_A receptors coupled with differences in the localization of inflammation-induced plasticity in Cl⁻ gradients (via changes in NKCC1 expression, localization or activity) could yield pharmacological profiles that account for the preservation of antinociceptive effects of midazolam observed in the presence of both inflammation and nerve injury (star 2).

NKCC1 transports Cl⁻ into the cell whereas KCC2 transports Cl⁻ out of the cell. In both cases, the process is electroneutral, i.e. there is no net charge transfer, and secondarily active, i.e. the co-transporters do not hydrolyze ATP themselves but instead rely on concentration gradients actively generated by the Na⁺/K⁺-ATPase (dotted arrows).

(A) Trace shows response to 20 ms-long puff of GABA onto a lamina I neuron from a P10 rat. Cartoon above shows the channel and the relative magnitude of Cl⁻ influx and HCO₃⁻ efflux. The net result is an outward current because the former is larger than the latter. (B) Trace shows response to 150 ms-long puff of GABA onto the same cell as in A. The current is initially outward but, over time, the Cl⁻ gradient collapses, thus reducing the magnitude of Cl⁻ influx until it eventually becomes smaller than HCO₃⁻ efflux. At that point, the current inverts and becomes inward. Modified from Coredero-Erausquin et al. (2005).

Data are based on same model described in Figure 2. (A) For E_anion = −70 mV, even modest inhibition reduced the slope of the input-output curve. Rate of inhibitory synaptic input (f_inh) is expressed relative to excitation, where α = f_inh/f_exc. For E_anion = −60 mV, reduction in potency of inhibition could be compensated by increased GABAergic transmission. For E_anion = −55 mV, this compensation failed (i.e. all dotted/dashed curves have roughly the same slope as the solid curve representing no inhibition). For E_anion = −50 mV, increasing GABAergic transmission paradoxically increased spiking. (B) Even if increasing GABAergic transmission managed to prevent frank disinhibition, the highly compensated system was prone to abrupt decompensation (i.e. small additional shifts in E_anion could cause substantial increases in firing). Graph here shows firing rate with inhibition (f_out) relative to firing rate without inhibition (f_out0) for different ratios of excitation to inhibition, as in A. Modified from Prescott et al. (2006).