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date: 26 April 2019

Regulation of Chloride Gradients and Neural Plasticity

Summary and Keywords

One of the most remarkable properties of neural circuits is the ability to restructure their synaptic connections throughout life. This synaptic plasticity allows neurons to structurally reorganize and adapt their function in response to experience. Among the multiple mechanisms that can modulate this property is synaptic inhibition by gamma-Aminobutyric acid (GABA) and/or glycine ionotropic receptors, which allow the flow of chloride and bicarbonate ions through the membrane. Neurons rely upon tight regulation of intracellular chloride for efficient inhibition through these receptors. The maintenance of chloride gradients is important not only to determine the strength of synaptic inhibition but also to determine its nature. Indeed, this inhibition can be hyperpolarizing or depolarizing, or with no outright change in the membrane potential. Despite the fact that membrane depolarization is commonly associated with excitation, depolarizing GABA/glycine can also produce inhibition, thereby highlighting the dual action of these neurotransmitters. Several considerations must be taken into account in order to allow depolarizing GABA/glycine responses to be excitatory. On the other hand, chloride homeostasis is never steady-state and even small changes of chloride across the membrane can impact the strength of inhibition. This dynamic effect has a direct impact on neuronal excitability and makes its regulation by changes in chloride gradients a highly tunable mechanism. Furthermore, increased excitability may also open a window for system refinement changes, such as synaptic plasticity. Indeed, the regulation of chloride homeostasis may underlie periods of enhanced plasticity, such as during early development. Finally, disruption of chloride gradients arises as a hub for pathology, which is evidenced in multiple disorders in the central nervous system.

Keywords: inhibition, GABA, glycine, KCC2, NKCC1, excitability, ionic plasticity

Introduction

The chloride ion (Cl) is one of the most abundant physiological anions in animal cells and is involved in important functions such as the control of cell volume, osmolarity, and pH balance, as well as cell proliferation and apoptosis (Delpire & Staley, 2014; Glykys et al., 2017; Kaila, Price, Payne, Puskarjov, & Voipio, 2014; Wilson & Mongin, 2018). The importance of Cl takes on a completely new significance in neurons, where it is involved in the regulation of cell excitability by synaptic inhibition. Evidently, the constraint on neuronal excitability is important to provide the balance between excitation and inhibition (E/I); however, inhibition is also important to produce effective oscillations in neural activity, as well as to regulate short- and long-term plasticity in neurons (Bartos, Vida, & Jonas, 2007; Fiumelli & Woodin, 2007; Lamsa, Kullmann, & Woodin, 2010; Somogyi, Katona, Klausberger, Lasztóczi, & Viney, 2014).

Fast synaptic inhibition is mediated primarily by ionotropic gamma-Aminobutyric acid (GABAA) and glycine receptors. Activation of these receptors opens an ion channel that enables the flux of anions across the membrane. The direction of the anion flux is dependent upon the electrochemical gradient for the anions that permeate though these receptors, which determines the driving force. The anion driving force can be easily computed as the difference between the resting membrane potential (Vm) and the anion reversal potential (Eanion):

Eanion=(RTF)ln([anion]i[anion]o),

where R is the perfect gas constant, T is absolute temperature, and F is the Faraday’s constant.

As Cl is the main anion that GABAA and glycine receptor channels are permeable to, EGABA/Gly is largely determined by ECl. However, bicarbonate anions (HCO3) also permeate through these receptor channels albeit to a lesser extent (1:4 ratio) (Kaila, 1994; Staley, Soldo, & Proctor, 1995). Therefore, EGABA/Gly is influenced by the transmembrane gradient and relative permeabilities for these two anions, as described by the Goldman-Hodgkin-Katz equation (Bormann et al., 1987):

EGABA/Gly=(RTF)ln(4[Cl]i+[HCO3]i4[Cl]o+[HCO3]o).

Regulation of Chloride Gradients and Neural PlasticityClick to view larger

Figure 1. Cl electrochemical gradient. Small changes in [Cl]i have a big impact in the direction of the Cl flow (Saraga, Balena, Wolansky, Dickson, & Woodin, 2008). Low [Cl]i make GABA responses hyperpolarizing (left). A small increase in [Cl]i produces no net anion flux through GABAA receptors (middle). Further increase in [Cl]i leads to depolarizing GABA (right). A depolarizing response does not mean that there is more Cl inside of the cell than outside; the anion flux (red arrows) depends on the electrochemical gradient, not just the concentration gradient.

When the intracellular concentration of chloride ([Cl]i) is higher than that dictated by the electrochemical gradient, ECl is positive relative to Vm. In this case, the opening of the GABAA/glycine channels results in efflux of Cl from the cell, thereby producing neuronal membrane depolarization. On the other hand, when [Cl]i is low, ECl is more negative than Vm, and GABAA/glycine receptor activation leads to Cl influx, which hyperpolarizes the neuron. Even a small change in [Cl]i can change the action of GABA/glycine from hyperpolarizing to depolarizing (Figure 1; Doyon et al., 2011; Prescott, Sejnowski, & De Koninck, 2006; Staley & Proctor, 1999). Most adult neurons in the central nervous system (CNS) maintain low [Cl]i to enable membrane hyperpolarization upon GABAA/glycine receptor activation. Indeed, the most recognized inhibitory action of GABA/glycine is by hyperpolarizing the cell membrane, which brings the membrane potential to more negative values and therefore increases the voltage gap to reach the threshold for action potential (AP) firing.

Membrane Hyperpolarization: Not the Sole Inhibitory Mechanism

Regulation of Chloride Gradients and Neural PlasticityClick to view larger

Figure 2. Shunting inhibition. In a classical cable model all transmembrane conductances are represented as a single resistor associated to a single battery. In this model, a shunting increase in conductance across the membrane (i.e., yielding no change in Vm) will decrease a concomitant EPSP akin to a short circuit effect (left). On the other hand, transmembrane conductances across the membrane can be more accurately described in an electro-diffusion model, because ion channels are selective for specific ion species and are therefore associated to different resistors (Doyon, Vinay, Prescott, & De Koninck, 2016). In this case, an excitatory current (Na+) is compensated for by a Cl current, at least partially, thereby decreasing the resulting depolarization. Contrary to the pure cable model, this shunting effect produces a significant Cl flux. E: potential; I: current. Red: outward current (Cl), green: inward current (Na+).

Opening of GABAA/glycine channels increases the permeability to Cl across the membrane, which then flows as dictated by Vm and ECl. This increase in Cl conductance is responsible for another inhibitory mechanism that has been referred to as shunting inhibition (Eccles, 1964). In the classical view, shunting refers to a divisive process, whereby an excitatory postsynaptic potential (EPSP) is reduced because of a short circuit as in a classical RC circuit (Figure 2; Doyon et al., 2016). Following this logic, it is thought that inhibition occurs with no net flux of Cl ions. However, the classical model does not take into account that each resistor (or conductor) in the circuit are specific to one ion. In a more accurate electro-diffusion model, one can see that under the shunting mode an excitatory current (carried by Na+ ions) is counterbalanced by the inhibitory current (carried by Cl) with no apparent hyperpolarization (Figure 2). That is, any deflection away from ECl is brought back by a Cl flux, keeping Vm from depolarizing. Indeed, even at times when the opening of GABAA/glycine channels increases the membrane conductance without producing any outright change in Vm (ECl = Vm and therefore, no driving force), an excitatory current will increase the driving force for Cl, which will make Cl flow again toward ECl, counteracting depolarization (Doyon et al., 2016). This “current shunt” effectively clamps Vm, thus reducing the ability of excitatory input to reach threshold and evoke an AP, hence inhibiting neuronal firing (Silver, 2010). Also, when ECl is positive to Vm but not enough to reach the AP firing threshold, GABA/glycine are depolarizing but can still produce inhibition by shunting (i.e., by clamping Vm away from threshold). An important corollary to this view is that in shunting mode, inhibition still involves a significant influx of Cl (Doyon et al., 2016; Prescott, 2015).

Channel Inactivation: Important for Presynaptic Inhibition

GABAA/glycine receptor-mediated depolarization can also produce inhibition via inactivation of voltage-gated Na+ and Ca2+ channels that are required for neurotransmitter release. In fact, shunting itself may not be enough to explain the inhibition produced by depolarizing GABA/glycine. Instead, the inactivation of Na+ and Ca2+ channels is probably more relevant to the inhibitory effect of GABA/glycine-mediated depolarization in presynaptic inhibition, representing another powerful means of reducing neuronal excitability in conditions where there is a high [Cl]i. (Bardoni et al., 2013; Graham & Redman, 1994; Rocha-Gonzalez, Mao, & Alvarez-Leefmans, 2008; Willis, 2006).

In conclusion, while loss of GABAA/glycine-mediated hyperpolarization clearly represents a form of disinhibition, depolarizing GABA/glycine currents do not necessarily mean that they produce direct excitation. Yet, in all of these cases, the Cl gradient remains an important determinant of the nature of the action of GABA/glycine ionotropic receptors on neuronal excitability and information transfer. Therefore, the regulation of Cl homeostasis may impact neuronal excitability and synaptic plasticity in multiple forms and may even be a unifying principle for pathologies with a characteristic imbalance between excitation and inhibition.

Defining Cl− Gradients

Prevalent Factors That Determine [Cl]i in Neurons

For years it was thought that Cl was distributed passively across the membrane, mostly based on observations from cells with highly-permeable membranes for this ion (Alvarez-Leefmans & Delpire, 2009). However, due to its participation in many important processes, it is clear that Cl must be tightly regulated. Most animal cells express a group of cation-chloride cotransporters (CCCs) that help establish the level of [Cl]i (Blaesse, Airaksinen, Rivera, & Kaila, 2009; Kaila, Price, et al., 2014; Payne, Rivera, Voipio, & Kaila, 2003). CCCs are membrane proteins capable of electroneutral secondary active transport, which exploit the transmembrane gradients for ionic species, for example Na+ and K+ that are set by the Na+/K+ ATPase. The CCC family includes four potassium–chloride cotransporters (KCC1–KCC4); two sodium–potassium–chloride cotransporters (NKCC1–NKCC2) and a sodium–chloride cotransporter (NCC). For neurons in the CNS, NKCC1 and KCC2 are of particular interest regarding the regulation of [Cl]i. As such, uptake of Cl is driven by NKCC1, which takes advantage of the plasmalemmal Na+ gradient and transports Cl ions along with Na+ and K+ ions into the cell. The stoichiometry for this process is generally thought to be 1Na+:1K+:2Cl (Russell, 2000). However, it has been suggested that it may be 1Na+:4K+:5 Cl instead, which yields an energetically more efficient transport (Brumback & Staley, 2008). On the other hand, KCC2 is the main extruder of Cl from neurons, also considered “neuron specific” as it is the only KCC not expressed in glial cells (Gagnon, Adragna, Fyffe, & Lauf, 2007; Payne, Stevenson, & Donaldson, 1996). It transports this ion against its chemical concentration gradient, using the outwardly directed K+ gradient. In addition, neurons express a battery of other Cl channels and transporters, including sodium-independent anion exchangers, which may also contribute to establish the basal [Cl]i level (Table 1).

Table 1. Proteins Involved in Chloride Homeostasis in Neurons

Protein

Gene

Type

Ion Species Involved

References

NKCC1

SLC12A2

CCC

Na+, K+, Cl

Arroyo, Kahle, & Gamba, 2013; Ben-Ari et al., 2012; Kaila, Price, et al., 2014; Wilson & Mongin, 2018

KCC1

SLC12A4

K+, Cl

Arroyo et al., 2013; Kaila, Price, et al., 2014; Wilson & Mongin, 2018

KCC2

SLC12A5

Arroyo et al., 2013; Chamma, Chevy, Poncer, & Levi, 2012; De Koninck, 2007; Kaila, Price, et al., 2014; Wilson & Mongin, 2018

KCC3

SLC12A6

Arroyo et al., 2013; Belenky et al., 2010; Kaila, Price, et al., 2014; Wilson & Mongin, 2018

KCC4

SLC12A7

Arroyo et al., 2013; Belenky et al., 2010; Kaila, Price, et al., 2014; Karadsheh, Byun, Mount, & Delpire, 2004; Wilson & Mongin, 2018

GABAAR

GABR

Cl-permeable ionotropic receptor

Cl > HCO3

Kaila, Price, et al., 2014; Wilson & Mongin, 2018; Yelhekar, Druzin, & Johansson, 2017

GLYR

GLR

Cl-permeable ionotropic receptor

Cl > HCO3

Kaila, Price, et al., 2014; Wilson & Mongin, 2018; Yelhekar et al., 2017

ClC-2

CLCN2

Voltage-dependent Cl channel

Cl

Ratté & Prescott, 2011; Rinke, Artmann, & Stein, 2010; Sík, Smith, & Freund, 2000; Smith, Clayton, Wilcox, Escudero, & Staley, 1995

ClC-3

CLCN3

Cl/H+ exchanger

Cl, H+

Stobrawa et al., 2001; Wilson & Mongin, 2018

ClC-5

CLCN5

Steinmeyer, Schwappach, Bens, Vandewalle, & Jentsch, 1995; Wilson & Mongin, 2018

ClC-6

CLCN6

Brandt & Jentsch, 1995; Poët et al., 2006; Wilson & Mongin, 2018

ClC-7

CLCN7

Brandt & Jentsch, 1995; Wilson & Mongin, 2018

ANO-1

TMEM16A

Ca+-activated Cl channel

Cl

Dauner, Lissmann, Jeridi, Frings, & Möhrlen, 2012; Vocke et al., 2013

ANO-2

TMEM16B

Cl

Dauner et al., 2012; Neureither et al., 2017; Vocke et al., 2013; Zhang et al., 2015

LRRC8A

LRRC8

VRAC

Cl, HCO3

Pedersen, Klausen, & Nilius, 2015; Voss et al., 2014

AE3

SLC4A3

Cl/HCO3 exchanger

Cl, HCO3

Romero, Chen, Parker, & Boron, 2013; Wilson & Mongin, 2018

NDCBE

SLC4A8

Na+-driven Cl/HCO3 exchanger

Cl, HCO3

Chen et al., 2008; Romero et al., 2000; Romero et al., 2013; Schwiening & Boron, 1994

NCBE

SLC4A10

Na+-activated Cl/HCO3 exchanger

HCO3

Parker et al., 2008; Romero et al., 2013; Wang, Yano, Nagashima, & Seino, 2000

CFTR

CFTR

ATP-phosphorylation- dependent anion channel

Cl

Krishnan, Maddox, Rodriguez, & Gleason, 2017

KBAT

SLC26A11

Voltage-dependent Cl-channel

Cl, HCO3, SO4

Rahmati et al., 2013; Rahmati et al., 2016; Rungta et al., 2015

Note. CCC = cation-chloride cotransporter; GABAAR = GABAA receptor; GLYR = glycine receptor; ClC = chloride channel; ANO = anoctamine; LRRC = leucine-rich volume-regulated anion channel; AE = anion exchanger; NCDBE = Na+-driven chloride bicarbonate exchanger; NCBE = Na+-driven chloride bicarbonate exchanger; CFTR = cystic fibrosis transmembrane conductance regulator; KBAT = kidney-brain anion transporter.

Most adult neurons maintain a low [Cl]i to enable hyperpolarizing inhibition, which is important to preserve the E/I balance. However, it has been argued that in early in development there is less need for hyperpolarizing inhibition because glutamatergic synapses have not yet formed. Accordingly, at these early stages Cl is actively accumulated in neurons and GABA acts as an excitatory neurotransmitter. The depolarizing action of GABA is required for neuronal plasticity in guiding neurogenesis and migration of young neurons (Ben-Ari, Gaiarsa, Tyzio, & Khazipov, 2007). As soon as glutamatergic synapses begin to form, there is a significant shift toward hyperpolarizing ECl (Rivera et al., 1999). This shift toward Cl extrusion has been associated with the differential expression of NKCC1 and KCC2 protein levels. Several transcription factors regulate the expression of these transporters across development. In early embryonic and postnatal stages, NKCC1 is highly expressed, while the expression levels of KCC2 are low (Ben-Ari, 2002). As maturation of the system takes place, there is an associated increased expression of KCC2 (Blaesse et al., 2006; Rivera et al., 1999; Uvarov et al., 2007).

Even then, protein expression does not correlate directly with the activity and function of KCC2/NKCC1. As for many other membrane proteins, these transporters are subjected to a number of intracellular regulatory mechanisms. Perhaps the best-known posttranslational modifications are phosphorylation/dephosphorylation mechanisms, but membrane recycling, protein cleavage, changes in quaternary structure (monomerization/oligomerization), and specific distribution within membrane domains have also been described, in particular for KCC2 (Kahle et al., 2013; Kaila, Price, et al., 2014; Medina et al., 2014; Tang, 2016). There is also accumulating evidence that KCC2 interacts with diverse proteins, which also regulate its function (Ivakine et al., 2013; Mahadevan et al., 2017). Regulatory pathways that affect KCC2 expression/function include brain derived neurotrophic factor (BDNF) signaling through its tyrosine-kinase receptor, tyrosine kinase B receptor (TrkB), 5-HT acting on 5HT2A receptors, Zn2+-dependent activation of metabotropic Zn2+ receptors, adenosine acting on its A3A receptors, as well as NMDA receptor activation by glutamate (Bos et al., 2013; Coull et al., 2005; Doyon et al., 2016; Lee et al., 2011; Mahadevan & Woodin, 2016; Puskarjov et al., 2015; Rivera et al., 2002).

The dynamic regulation of NKCC1 and KCC2 proteins, critical for determining the direction of GABA-mediated responses, does not occur exclusively during early development. Even in mature neurons, the levels of KCC2 appear to be regulated by diverse transcriptional and posttranslational factors. The multiple regulatory mechanisms that decrease or enhance KCC2 activity ultimately define the nature of GABAA/glycine receptor-mediated responses (Tang, 2016). Accordingly, alterations of KCC2 function can increase [Cl]i thereby weakening the electrochemical Cl gradient and disrupting synaptic inhibition. Also, as shunting inhibition occurs to counteract excitatory input, it also involves a significant Cl influx, such that it is liable to failure by the depletion of the Cl gradient (Doyon et al., 2016; Prescott, 2015; Prescott et al., 2006). Reduced KCC2 function has been implicated in many CNS conditions and disorders characterized by an impaired E/I balance, including epilepsy, stress, motor spasticity, schizophrenia, autism, and chronic pain (Boulenguez et al., 2010; Coull et al., 2003; Hewitt et al., 2009; Hyde et al., 2011; Merner et al., 2015). Nevertheless, a reduced level of KCC2 activity is not exclusive to pathological states. Even in physiological conditions, the levels of KCC2 and [Cl]i vary among cell populations and can lead to cell-type-specific differences in EGABA/Gly (Chavas & Marty, 2003; Grob & Mouginot, 2005; Gulácsi et al., 2003; Martina, Royer, & Pare, 2001). These differences in Cl extrusion capacity do not necessarily produce an outright change in the polarity of GABA/glycinerergic currents, but they may affect the spatial and temporal summation of inhibitory inputs, especially during increased synaptic activity (Cordero-Erausquin, Coull, Boudreau, Rolland, & De Koninck, 2005; Grob & Mouginot, 2005; Hewitt et al., 2009).

Activity-Dependent Cl−-Loading in Neurons

Regulation of Chloride Gradients and Neural PlasticityClick to view larger

Figure 3. Activity-dependent collapse of inhibition. In normal conditions, KCC2 is able to effectively extrude Cl to maintain inhibitory activity upon repetitive input (left). In conditions with low KCC2 there is a larger depression of the inhibitory events at a holding potential of 0 mV due to an intracellular accumulation of Cl flowing in through GABAA channels (right). At a holding potential of −80 mV, when there is a dominant HCO3 outflux, little collapse in GABAA current occurs because HCO3 is continuously restored by the action of a carbonic anhydrase enzyme (Ferrini et al., 2013; Hewitt, Wamsteeker, Kurz, & Bains, 2009; Kaila et al., 1990).

Regulation of Chloride Gradients and Neural PlasticityClick to view larger

Figure 4. Depolarizing GABA. (A) NKCC1-dependent Cl loading produces a depolarizing EGABA. Neurons in early development and primary afferents are examples of cells that express negligible KCC2 but high levels of NKCC1, which yield depolarizing GABAA responses. Carbonic anhydrase expression is low during early development, thus EGABA typically equals ECl and can be calculated by the Nernst equation. (B) KCC2-mediated extrusion enables hyperpolarizing inhibition. If EGABA depended only on Cl, blockade of KCC2 would redistribute Cl across the membrane until a newly found equilibrium at Vm (left). GABA in these conditions would produce no overt depolarization nor hyperpolarization. Depolarizing GABA arises from the partial permeability to HCO3. EGABA is given by the Goldman-Hodgkin-Katz equation. Loss of Cl extrusion brings ECl to Vm but as EHCO3 is more depolarized, EGABA is more depolarized than Vm (right) (Bormann, Hamill, & Sakmann, 1987).

High levels of synaptic activity are not a rare occurrence in neural circuits. Neurons are not silent, isolated entities but are constantly undergoing continuous synaptic activity (Destexhe & Pare, 1999). As such, a neuron is subject to both glutamatergic and GABA/glycinergic inputs. Glutamatergic activity produces a strong depolarization of the neuronal membrane that brings the Vm toward the firing threshold. This positive value of Vm increases the driving force for Cl (Doyon et al., 2011). Therefore, [Cl]i is also strongly influenced by the opening of Cl channels, such as through the activation of GABAA/glycine receptors (Cordero-Erausquin et al., 2005; Staley et al., 1995). In this case, the activity of KCC2 is important to replenish the Cl gradient to maintain effective inhibition, albeit at a lower rate than fast synaptic events. Importantly, because KCC2 is electroneutral, it serves to clear Cl accumulation from its influx through GABAA/glycine receptor channels without causing depolarization, thus acting as an electrically silent homeostatic mechanism. Prolonged activation of such receptors can lead to a large Cl influx such that Cl cannot be extruded by KCC2 fast enough, leading to a collapse of the Cl gradient (Figure 3; Cordero-Erausquin et al., 2005; Doyon et al., 2011; Doyon et al., 2016; Ferrini et al., 2013; Hewitt et al., 2009). This decrease in synaptic strength due to a reduced Cl gradient is sometimes termed ionic plasticity (Kaila, Ruusuvuori, Seja, Voipio, & Puskarjov, 2014; Raimondo, Markram, & Akerman, 2012; Rivera, Voipio, & Kaila, 2005). Such dynamic failure of synaptic inhibition is compounded in neurons with a constitutively lower expression of KCC2 (Doyon et al., 2011; Grob & Mouginot, 2005) and in pathological conditions involving loss of KCC2 function (Boulenguez et al., 2010; Coull et al., 2003; Ferrini et al., 2013; Hewitt et al., 2009).

In certain cases, KCC2 hypofunction can even lead to a shift in the polarity of inhibitory currents from hyperpolarizing to depolarizing (Cordero-Erausquin et al., 2005; Grob & Mouginot, 2005; Hewitt et al., 2009). In absence of inward Cl transport (through NKCC1, for example; Figure 4A), depolarizing GABA cannot be explained by a depolarizing ECl. Indeed, because KCC2 is a passive transporter, loss of Cl extrusion will cause [Cl]i to accumulate until ECl reaches Vm, but not further (Figure 4B). The depolarization results from the outflux of HCO3 that also permeates through GABAA/glycine receptors, because EHCO3 is more depolarized (Figure 4B) (~ −10 mV). After the resulting collapse in Cl gradient, the remaining HCO3 current predominates and causes Vm to move toward the depolarized EHCO3 (Staley et al., 1995). This type of overt depolarization may be responsible for the paradoxical responses observed by benzodiazepine administration in young animals, as well as catastrophic seizures in epileptic conditions (Cohen, Navarro, Clemenceau, Baulac, & Miles, 2002; Staley et al., 1995).

Reciprocal Influence of [Cl]i and GABAA Receptors

The influence of GABAergic activity and [Cl]i goes both ways. Activation of GABAA receptors temporarily produce Cl loading into cells, which lead to changes in [Cl]i. These differences in [Cl]i can, in turn, alter the expression of GABAAR subunits (Succol, Fiumelli, Benfenati, Cancedda, & Barberis, 2012). The subunit composition of GABAA receptors determines their pharmacological and biophysical properties, as well as the subcellular localization of each receptor (synaptic, perisynaptic, or extrasynaptic) (Farrant & Nusser, 2005; Olsen & Sieghart, 2008). These characteristics are important to define whether a GABAA receptor is more likely to participate in fast, transient inhibition or in a form of persistent “tonic” inhibition. As such, Cl gradients may modulate the nature of GABAergic transmission, and this transmission may then affect how Cl is dynamically loaded into neurons. However, it may be difficult to establish the causality, because these variables are constantly interacting. In the end, the only way to understand the function of this complex interplay may be to measure the overall effect on the net output of the network.

Besides GABA/glycine-activated ionotropic receptors, Ca2+-dependent and voltage-dependent Cl channels can provide additional Cl fluxes during neuronal activity. These channels can also transiently change [Cl]i. For example, anoctamine channels are Ca2+-activated Cl channels that can therefore modulate the efficacy of inhibitory synapses by increasing postsynaptic Cl concentration (Neureither, Ziegler, Pitzer, Frings, & Mohrlen, 2017; Zhang, Schmelzeisen, Parthier, Frings, & Mohrlen, 2015). On the other hand, voltage-sensitive Cl channels, such as ClC-2, may also contribute to transient changes in [Cl]i. ClC-2 mediates inward-rectifying currents that have been proposed to mediate Cl efflux after a rise in [Cl]i (Földy, Lee, Morgan, & Soltesz, 2010; Staley, 1994). This is indeed the case when ECl is less negative than Vm,; however, it has been argued that since Cl follows its electrochemical gradient, ClC-2 channels normally leak Cl into the cell rather than out (Ratté & Prescott, 2011).

Subcellular Cl− Domains

Cl loading through activation of Cl channels would be expected to be more pronounced in smaller cell compartments such as the restricted area of distal dendrites and spines. Indeed, dendrites experience rapid Cl accumulation during persistent activity, leading to an intracellular gradient of Cl distribution (Doyon et al., 2011). However, other differences in subcellular [Cl]i may be hard to explain solely by the expression levels of CCCs because Cl is a highly mobile ion and diffuses rapidly (Delpire & Staley, 2014). A transient Cl load at a GABAA synapse will influence the local EGABA but may also spread to neighboring synapses, especially with low levels of KCC2 (Doyon et al., 2011). Therefore, a standing [Cl]i gradient arises from the combined action of KCC2 activity and background synaptic GABAAR activity, as KCC2 appears to be distributed uniformly across the membrane (Doyon et al., 2011).

A complementary means to maintain different Cl gradients may arise from the relative concentration of impermeant anions inside ([A]i) and outside ([A]o) the cell, which produce a Gibbs-Donnan effect that limits the mobility of Cl (Glykys et al., 2014). The [A]i arises from negatively charged phosphate groups that make up the DNA backbone and proteins with negatively charged amino and carboxyl groups. On the other hand, the extracellular matrix (ECM) contains negatively charged sulfates on proteoglycans that make up most of [A]o. Therefore, Cl is not distributed evenly across the membrane due to the constraint of negative impermeant anions to maintain a Gibbs-Donnan equilibrium. In fact, ECM also displays developmental shifts during maturation, and its dissolution can redistribute Cl across the membrane, leading to an increase in [Cl]i in adult neurons (Glykys et al., 2014).

Importantly, the increased permeability to Cl by the activation of Cl channels can drive EGABA away from the Gibbs-Donnan equilibrium dictated by impermeant anions. Normal EGABA can then be restored by the activity of CCCs, such as KCC2. Therefore, impermeant anions may be necessary for establishing the basal Cl levels in neurons, but CCCs appear important to compensate for activity-dependent changes in Cl (Doyon et al., 2016). Indeed, the multiple means of KCC2 regulation by transcriptional and posttranslational factors makes this a highly tunable mechanism (Kahle et al., 2013; Kaila, Price, et al., 2014; Medina et al., 2014; Tang, 2016).

The Impact of Cl− Gradients on Neuronal Excitability

Besides important functions such as cell volume regulation, in neurons, the most striking role of Cl is the control of cell excitability (Raimondo, Richards, & Woodin, 2017). However, Cl is probably not the first ion that comes to mind when one refers to changes in neuronal excitability. The role of Cl in this process is perhaps less straightforward than the effect of tampering with Na+ or K+ channels, which impact directly the generation of APs. Disrupting cell firing in this way is an all-or-nothing response and gives little room for adjustments. In contrast, the distribution of Cl across the membrane is actively regulated and provides for a wide range of tuning opportunities. Even small changes in [Cl]i can lead to a difference in EGABA/Gly and therefore in the strength of inhibition. In this way, it is easy to see how changing Cl gradients would lead to a change in neuronal excitability.

Is Depolarizing GABA Excitatory?

A depolarizing EGABA/Gly, does not correlate directly with AP firing. As outlined previously, Cl conductances can produce shunting inhibition or “clamp” voltage changes even in depolarizing conditions. Also, prolonged GABAA depolarization can cause Na+ channel inactivation, which also produces inhibition (Graham & Redman, 1994). Nevertheless, depolarization does bring Vm closer to the firing threshold, thereby also potentially increasing the probability of AP firing (Takkala, Zhu, & Prescott, 2016). Many factors must be considered in making GABA/glycine excitatory. First, depolarizing GABA is due to HCO3, not Cl. During repetitive GABAergic activation, there can be considerable Cl accumulation, until the Cl gradient is exhausted and ECl approaches Vm. The HCO3 flux, however, is actively maintained through the continuous action of a carbonic anhydrase enzyme (Kaila, Saarikoski, & Voipio, 1990; Staley et al., 1995). This flux brings Vm toward the very depolarized EHCO3. Indeed, increased neuronal excitability can occur after the Cl gradient collapses (Hewitt et al., 2009). Although this mechanism does not necessarily involve Cl efflux, it does require sufficient degradation of its gradient (Prescott, 2015).

A second important consideration for excitatory GABA is the subcellular location of inhibitory synapses (Gulledge & Stuart, 2003; Jean-Xavier, Mentis, O’Donovan, Cattaert, & Vinay, 2007). Indeed, a depolarizing GABA response at the soma would inhibit an EPSP that occurs nearby, because of the local the shunting effect. On the other hand, a depolarizing GABA response in a dendrite is excitatory, because the depolarization, but not the shunt, propagates to the soma and can sum with an EPSP to produce AP firing. In addition, depolarization kinetics could also contribute to make GABA excitatory as fast depolarizations may increase excitability, but slow depolarizations may produce Na+ channel inactivation (Takkala et al., 2016).

Third, little Cl is needed to produce these excitatory effects. It has been suggested that a mere rise of 2.5 mM in [Cl]i can lead to increased firing rates, as well as an increased number of spikes, which affect the output of the neuron (Saraga et al., 2008). A similar observation was made when using an optogenetic chloride pump, halorhodopsin (NpHR) to artificially load Cl into neurons; NpHR-mediated Cl loading also shifts EGABA/Gly, and produces a transient shift in network excitability (Alfonsa et al., 2015; Raimondo, Kay, Ellender, & Akerman, 2012). Interestingly, dissolution of the ECM also produces Cl accumulation inside the cells and increases excitability, further supporting the role of the ECM in Cl homeostasis (Balmer, 2016).

The depolarizing effects of GABA, either due to activity-dependent collapse in ECl or active Cl loading, can be reduced with carbonic anhydrase inhibitors (which degrade the depolarizing HCO3 gradient) or with blockers of Cl import (e.g., the NKCC1 inhibitor bumetanide), which have been reported to have anticonvulsant and analgesic actions (Asiedu, Mejia, Hubner, Kaila, & Price, 2014; Bruno et al., 2016; Ferrini et al., 2013; Thiry, Dogné, Supuran, & Masereel, 2007).

KCC2: A Bidirectional Transporter

Cell spiking also increases the driving force for Cl, thereby producing additional Cl loading (Doyon et al., 2011). This effect is especially important when KCC2 expression or activity is altered, such that it does not produce effective Cl extrusion. Indeed, KCC2 blockade can produce intracellular accumulation of Cl and depolarized ECl (Rivera et al., 1999; Woodin, Ganguly, & Poo, 2003). Moreover, it has been suggested that enhanced Cl loading can change the driving force for KCC2 extrusion, because its activity depends on the energy stored in the chemical concentration gradients of both Cl and K+ (Delpire & Lauf, 1991). This change would produce a switch in the direction of KCC2 transport, which would lead to an increase in [K]o and further cell depolarization (Sipila, Huttu, Soltesz, Voipio, & Kaila, 2005; Viitanen, Ruusuvuori, Kaila, & Voipio, 2010). However, KCC2 appears to have a stronger impact on effective inhibition, thereby decreasing neuronal firing (Doyon et al., 2011). As such, the net effect of increased KCC2 activity limits [K]o accumulation by maintaining the excitability of the network at bay.

The amount of [Cl]i that could produce a reversal of KCC2 activity is beyond normal physiological conditions (Doyon et al., 2011). However, these conditions could be met in pathological states, where there is an excess loading of Cl into neurons, most notably in epilepsy (Alfonsa et al., 2015). In fact, these disorders are often found in conjunction with a concomitant decrease in KCC2 (Table 2). Therefore, in hyperactive states (e.g., epilepsy), the increased firing would lead to a K+ accumulation outside and further Cl loading, because the direction of the K+/Cl flow by KCC2 is also dependent on the K+ gradient (Balena, Acton, Koval, & Woodin, 2008). The possibility of reversing the direction of KCC2 by increasing [K]o is useful to measure KCC2 activity (Ferrini et al., 2013; Gagnon et al., 2013; Taylor et al., 2016). However, whether KCC2 really imports Cl in physiological or even in pathological conditions is not yet clear.

On the other hand, a decrease in [Cl]i enhancing GABA-mediated hyperpolarization could also affect neuronal excitability, for example, through activation of voltage-gated channels sensitive to hyperpolarization (Bonin, Zurek, Yu, Bayliss, & Orser, 2013). This is the case of hyperpolarization-activated cyclic nucleotide-gated channels, which promote rebound spiking by producing a depolarizing Ih current (He, Chen, Li, & Hu, 2014). This effect is particularly important in conjunction with the associated Cl load, rather than hyperpolarization alone, thereby highlighting the importance of Cl gradients in regulating neuronal excitability (Madisen et al., 2012; Raimondo, Kay, et al., 2012; Tønnesen, Sørensen, Deisseroth, Lundberg, & Kokaia, 2009).

From Changes in Excitability to Synaptic Plasticity

Transient changes in neuronal excitability can occur after a temporary depolarization of ECl and a consequent weakening of inhibitory strength, which are typically restored by the action of KCC2 (Kaila, Ruusuvuori, et al., 2014; Moore, Kelley, Brandon, Deeb, & Moss, 2017). Transient as they may be, these changes in excitability and inhibition may also open a window for the induction of system refinement changes, such as synaptic plasticity. Indeed, neurons modify their synaptic connections in response to experience, and this enables adjustments to the environment. This ability is present throughout life, but there are certain periods when there is an increased capacity to restructure connections (Berardi, Pizzorusso, & Maffei, 2000). These periods of enhanced plasticity often occur during early development. Normally, it is assumed that maintaining the E/I balance is important because too much inhibition may prevent plasticity whereas exaggerated excitation may lead to excitotoxicity. Yet, depolarizing GABA at early developmental stages appears important to favor enhanced plasticity and to achieve neurogenesis, migration, synaptogenesis, and network formation, for example. It has been argued that this depolarizing GABA may be more failsafe than glutamatergic transmission because it provides a more limited level of depolarization than glutamatergic mechanisms (Ben-Ari, 2002).

Cl− Regulation and Early Life Plasticity

Interestingly, ECM and CCC proteins undergo significant developmental shifts that accompany a gradual change in Cl distribution across the membrane (Ben-Ari, Khalilov, Kahle, & Cherubini, 2012; Frischknecht et al., 2009). These changes in Cl homeostasis may underlie synaptic refinement during development. Indeed, in early life stages ECl is depolarized with respect to Vm and GABA behaves as a depolarizing neurotransmitter (Ben-Ari, 2002). Depolarizing GABA at this stage is important to guide neurogenesis and migration of young neurons. Because ECl is depolarized, GABA is able to increase intracellular Ca2+ and generate primitive oscillations that are fundamental for network activity (Ben-Ari et al., 2007). These depolarizations are also important to activate NMDA receptors that are expressed at silent synapses in the immature brain (Isaac, Crair, Nicoll, & Malenka, 1997; Leinekugel, Medina, Khalilov, Ben-Ari, & Khazipov, 1997; Wang & Kriegstein, 2008). At resting membrane potential, there is no ion flux through NMDA receptors because their channel is blocked by extracellular Mg2+ and require a postsynaptic depolarization to remove it. These depolarizations are important for insertion of AMPA receptors at functional synapses (Liao & Malinow, 1996; Wang & Kriegstein, 2011). On the other hand, even if GABA-induced depolarizations can be excitatory, they can also decrease the effectiveness of excitatory neurotransmission by clamping the membrane to ECl through membrane shunting. This way, GABA can exert dual action mediating excitation and inhibition in immature neurons. Once glutamate begins to act as the main excitatory transmitter, the shift of ECl appears to occur to enable hyperpolarizing inhibition (Ben-Ari, 2002; Ben-Ari et al., 2012).

The Missing Link: BDNF

A critical contender in the transition from depolarizing to hyperpolarizing GABA is BDNF. BDNF is a neurotrophin with important effects in neuronal survival and differentiation, as well as synapse formation and plasticity (Park & Poo, 2013). Signaling of BDNF through its receptor TrkB has a role in the regulation of Cl regulation by promoting the upregulation of KCC2 expression in young neurons (Aguado et al., 2003; Ludwig et al., 2011). Also, BDNF can be released from dendrites in an activity-dependent manner (Kuczewski, Langlois, et al., 2008; Kuczewski, Porcher, et al., 2008). The fact that BDNF can modulate KCC2 expression suggests that there can be activity-dependent modulation of Cl homeostasis. In immature neurons BDNF potentiates GABAergic activity, thereby promoting activity-dependent plasticity though GABA-induced Ca2+ elevations (Porcher et al., 2011).

Synaptic Plasticity in Mature Neurons

Even in adult neurons, synapses can be modified in structure and function in response to external stimuli and experience. This plastic capacity is thought to be the basis for different physiological processes such as learning and memory, as well as pathological chronic pain. At glutamatergic synapses, the rate of activity can produce short- or long-term changes in the efficiency of synaptic transmission, such as long-term potentiation and depression (LTP/LTD) and spike-timing dependent plasticity (Bear & Malenka, 1994; Bliss & Collingridge, 1993; Lamsa et al., 2010). Here too, membrane depolarization is important to release the Mg2+ block from NMDA receptors. These depolarizations are normally associated with AMPA receptor activation; however, they can also occur though excess GABAergic activity, which enables Ca2+ influx and plasticity (Gubellini, Ben-Ari, & Gaiarsa, 2001; Leinekugel et al., 1997; Schmanns & Friauf, 1994; Sipila et al., 2005). This is especially true during large spontaneous activity because glutamatergic signaling can downregulate KCC2 (Kitamura et al., 2008; Lee et al., 2011; Puskarjov, Ahmad, Kaila, & Blaesse, 2012; Rivera et al., 2004; Toyoda et al., 2003; Zhou et al., 2012). In particular, theta-burst stimulation (TBS), such as that used in the hippocampus to produce synaptic plasticity, can induce a shift in EGABA/Gly, which is dependent on KCC2 activity (Wang, Gong, & Xu, 2006). In fact, following TBS there is an activity-dependent decrease in KCC2 and a depolarizing EGABA/Gly that are also the cause for the spreading of potentiation to unstimulated synapses (Ferando et al., 2016). Therefore, low levels of KCC2 may be particularly sensitive to this effect.

The mechanisms that underlie activity-dependent plasticity are also mediated by BDNF (Brady et al., 2017; Grau & Huang, 2018; Lu, Park, & Poo, 2014). As BDNF regulates synaptic plasticity during development, it is not surprising that it does so too in the adult. The effect of BDNF on GABAergic neurotransmission seems to be regulated in parallel with the shift in ECl. Therefore, when GABA is inhibitory in mature neurons, BDNF decreases GABAergic transmission. Also, in these late stages, BDNF produces KCC2 downregulation, rather than upregulation (Rivera et al., 2002). This differential effect is explained by different signaling pathways that are activated by BDNF-activated TrkB receptors in young and adult neurons (Ferrini & De Koninck, 2013). KCC2 downregulation also enables NMDA receptor potentiation by BDNF, thereby highlighting the role of this neurotrophin in synaptic plasticity (Hildebrand et al., 2016).

Inhibitory Plasticity

GABAergic synapses also express multiple forms of synaptic plasticity (Maffei et al., 2017). Indeed, because they act through Cl influx, GABAA currents themselves can tune the strength of inhibition by progressively causing Cl accumulation, which collapses hyperpolarizing inhibition. This transient ionic plasticity is unique to GABAergic currents and causes a form of short-term disinhibition that may be important for expression of long-term synaptic plasticity at excitatory synapses (Hildebrand et al., 2016).

On the other hand, coincident synaptic stimulation has been shown to induce LTD at GABAergic synapses through a long-term depolarizing shift in EGABA, weakening synaptic inhibition (Ormond & Woodin, 2009, 2011; Woodin et al., 2003). This effect is mediated by a decrease in KCC2 function (Woodin et al., 2003).

Maladaptive Plasticity From Cl− Dysregulation

Plastic modifications can convey changes in neural firing in the long term. Indeed, it is not only excitability that enables synaptic plasticity but also synaptic plasticity that can explain changes in neuronal firing. After all, it is not the plasticity itself but how the activity of the synapse modifies a neuronal response that underlies a behavioral phenotype (Carvalho & Buonomano, 2009). In this regard, Cl gradients are important to enable network modifications, and their dysfunction may underlie the noisy and unstable neuronal networks that are present in several behavioral and cognitive deficits. In fact, more and more conditions and pathologies have been associated with altered Cl gradients (Table 2). These could be regarded as yet another form of plasticity, perhaps just maladaptive.

Table 2. Cl− Dysregulation in Disease

Disease

Protein

Alteration

Defective Dysfunction

Selected References

ALS

KCC2

Reduced expression/activity

Hyperexcitability

Fuchs et al., 2010 ; Lederer, Torrisi, Pantelidou, Santama, & Cavallaro, 2007

No change

Mòdol, Mancuso, Alé, Francos-Quijorna, & Navarro, 2014

NKCC1

No change

Fuchs et al., 2010

Alzheimer’s disease

KCC2

APP-KCC2 interaction

Reduced inhibition

Chen et al., 2017; Doshina et al., 2017

Reduced GABA-induced hyperpolarization

NKCC1

No change

Doshina et al., 2017

GABAAR

GABAAR agonists/antagonists

Decreased Aβ‎ generation

Li, Sun, et al., 2016

Improved memory and cognition

Andermann syndrome

KCC3

Reduced expression

Volume control

Ding & Delpire, 2014; Rudnik-Schöneborn et al., 2009; Shekarabi et al., 2012

ACC

ASD

KCC2

Reduced expression/activity

Depolarized EGABA

Banerjee et al., 2016; Duarte et al., 2013; Merner et al., 2015; Tang et al., 2016

Delayed functional E/I switch

Increased NKCC1/KCC2 ratio

Behavior deficits

Familial febrile seizures

NKCC1

Increased expression/activity

Depolarized EGABA

Tyzio et al., 2014

Autistic-like-behavior

GABAAR

Decreased expression

Decreased BZD binding

Adusei, Pacey, Chen, & Hampson, 2010; Cellot & Cherubini, 2014; Coghlan et al., 2012

Mutations

Decreased social interaction

Subunit downregulation

Hyperexcitability

Chronic pain

KCC2

Reduced expression/activity

Depolarized EGABA

Coull et al., 2003; Coull et al., 2005; Hasbargen et al., 2010; Kahle, Khanna, Clapham, & Woolf, 2014; Sanchez-Brualla et al., 2017

Enhanced activity

Hyperalgesia/allodynia

NKCC1

Increased expression/activity

Depolarizing EGABA shift in afferents

Galan & Cervero, 2005; Pitcher & Cervero, 2010; Pitcher, Price, Entrena, & Cervero, 2007; Willis, 1999

DRR

Mechanical hyperalgesia

Visceral pain

No change

Nomura, Sakai, Nagano, Umino, & Suzuki, 2006; Zhang, Liu, & Xu, 2008

GABAAR

GABAAR antagonists

Enhanced activity Hyperalgesia/allodynia

Loomis, Khandwala, Osmond, & Hefferan, 2001; Sivilotti & Woolf, 1994; Yaksh, 1989

GLYR

GLYR antagonists

Enhanced activity

Loomis et al., 2001; Sherman & Loomis, 1994; Sivilotti & Woolf, 1994

Hyperalgesia/allodynia

Down syndrome

NKCC1

Increased expression/activity

Depolarized EGABA

Deidda et al., 2015

Impaired synaptic plasticity

Memory deficits

Drug dependence

KCC2

Decreased expression/activity

Impaired Cl extrusion

Taylor et al., 2016; Thomas et al., 2018

Depolarized EGABA

Depressed reward circuit

Edema

KCC2/

KCC3

Reversed activity

KCl loading

DeFazio, Keros, Quick, & Hablitz, 2000; Glykys et al., 2017; Payne, 1997

Cell swelling

Epilepsy

KCC2

Reduced expression/activity

Depolarized EGABA

Buchin, Chizhov, Huberfeld, Miles, & Gutkin, 2016; Campbell et al., 2015; Huberfeld et al., 2007; Kahle, Merner, et al., 2014; Kahle et al., 2016; Khalilov et al., 2011; Li et al., 2008

Increased spontaneous activity

Seizures

KCC3

Reduced expression

Cell swelling

Boettger et al., 2003

Hyperexcitability

NKCC1

Increased activity

Proconvulsant

Dzhala et al., 2005; Dzhala et al., 2010; Li et al., 2008; Zhu, Polley, Mathews, & Delpire, 2008

Synchronous bursts of APs

Epileptiform activity

ClC-2

Increased channel dynamics

Hyperexcitability

Ratté & Prescott, 2011; Saint-Martin et al., 2009

Huntington’s disease

KCC2

Htt-KCC2 interaction

Depolarized EGABA

Dargaei et al., 2018

Altered synaptic plasticity

Reduced expression

Memory deficits

NKCC1

Increased expression

Depolarized EGABA

Dargaei et al., 2018

Altered synaptic plasticity

Memory deficits

Hyperekplexia

GLYRs

Reduced expression

Altered synaptic inhibition

Demir et al., 2014; Schaefer, Langlhofer Kluck, & Villmann, 2013; Xiong et al., 2014

Ischemic stroke

KCC2

Reduced expression/activity

Reduced RDD

Toda, Ishida, Kiyama, Yamashita, & Lee, 2014

Motor spasticity

MIH

KCC2

Reduced expression/activity

Depolarized EGABA

Ferrini et al., 2013; Ferrini, Lorenzo, Godin, Quang, & De Koninck, 2017

Parkinson disease

NKCC1

Dopamine-deprivation-dependent raise in [Cl]i

Impaired E/I balance

Lozovaya et al., 2018

Enhanced activity

Motor deficiency

Schizophrenia

KCC2

Reduced expression/activity

Familial febrile seizures

Arion & Lewis, 2011; Hyde et al., 2011; Merner et al., 2015; Sullivan, Funk, Shan, Haroutunian, & McCullumsmith, 2015

Psychotic episodes

NKCC1

Increased expression/activity

Psychotic episodes

Arion & Lewis, 2011; Hyde et al., 2011; Merner et al., 2016

SCI

KCC2

Reduced expression/activity

Depolarized EGABA

Boulenguez et al., 2010; Bos et al., 2013; Liabeuf et al., 2017

Reduced RDD

Motor spasticity

Stress

KCC2

Reduced expression/activity

Depolarized EGABA

Hewitt et al., 2009; Ostroumov et al., 2016

Collapsible Cl gradient

Increased alcohol self-administration

Note. ACC = agenesis of corpus callosum; ALS = amyotrophic lateral disease; AP = action potential; APP = amyloid precursor protein; ASD = autism spectrum disorders; BZD = benzodiazepine; DRR = dorsal root reflexes; Htt = huntingtin protein; MIH = morphine-induced hyperalgesia; RDD = rate-dependent depression of the Hoffman reflex; SCI = spinal cord injury.

Energetic Dilemma

A common observation in the aforementioned conditions (Table 2) is KCC2 downregulation and an associated impaired inhibition by Cl accumulation. This effect, however, has been proposed to be a compromise between disinhibition and a means to preserve energy in the otherwise energy-burning situations, such as epileptic seizures (Bahar, Suh, Zhao, & Schwartz, 2006; Du, Li, Wang, & Wu, 2016; Ingram, Zhang, Xu, & Schiff, 2013). In hyperactive states, the probability that there will be coincident excitatory and inhibitory input onto a neuron is enhanced, thereby increasing the cost of both these systems (Buzsáki, Kaila, & Raichle, 2007). This temporal overlap in contrasting activity would produce shunting but also increased Cl loading, as described previously. In these conditions, KCC2 would be working at a high rate to keep a low [Cl]i level, thereby exploiting the K+ gradient set by the Na+/K+ ATPase. Decreasing the effective export of Cl would reduce the driving force of the ion fluxes, thereby removing an energetic constraint on homeostasis (Buzsáki et al., 2007; Kaila, Ruusuvuori, et al., 2014).

Catastrophic Collapse of Inhibition

An important downregulation of KCC2 may critically impact the strength of inhibition. Indeed, an impaired inhibition by Cl accumulation may lead to abnormal activity due to enhanced excitability and synaptic plasticity. In particular, the depolarizing EGABA may activate NMDA receptors by releasing their Mg2+ block, as well as enabling the potentiation of these receptors by BDNF (Hildebrand et al., 2016; Li, Chen, et al., 2016). NMDA receptor activation, in turn, may produce a further downregulation of KCC2 in an activity-dependent manner (Lee et al., 2011; Puskarjov et al., 2012). Therefore, this positive feedback loop may carry the potential for a complete collapse of inhibition and the possibility of catastrophic events (Figure 5A).

Regulation of Chloride Gradients and Neural PlasticityClick to view larger

Figure 5. Catastrophic collapse of inhibition. (A) Downregulation of KCC2 can lead to depolarizing GABAA/glycinergic responses due to Cl accumulation. This depolarization can thereby release the Mg2+ block from NMDA receptors, as well as allow the potentiation of these receptors by Fyn-mediated phosphorylation, causing cation flux (Hildebrand et al., 2016; Li, Chen, et al., 2016). NMDA receptor activation causes KCC2 downregulation through activation of proteases, which translates into a positive-feedback loop for disinhibition (Lee, Deeb, Walker, Davies, & Moss, 2011; Puskarjov et al., 2012). At a short-term scale, Cl accumulation causes membrane depolarization, which increases the driving force for more Cl accumulation. This effect can lead to enhanced firing by short-term disinhibition. KCC2 downregulation can cause bistability, transforming normal spiking into bursting activity (B; Doyon, Prescott, & De Koninck, 2015), loss of LTP specificity, thereby spreading to neighboring synapses (C; Ferando, Faas, & Mody, 2016), and signal crosstalk between normally separate pathways (D; Keller, Beggs, Salter, & De Koninck, 2007; Lavertu et al., 2014).

One example of abnormal excitability is the synchronous and excessive neuronal activity during epileptic seizures. Indeed, depolarizing GABAergic synaptic events due to Cl accumulation are thought to prime neurons for interictal-like and seizure events associated with epilepsy (Alfonsa et al., 2015). Also, weak but sustained excitatory events that do not elicit spiking under normal Cl homeostasis conditions can be detected when KCC2 is reduced, even producing abnormal busting of APs (Figure 5B; Doyon et al., 2015).

Degradation of Information Transfer

Furthermore, compromised inhibition can affect the fidelity of information transfer. This effect has been explored using computer simulations, where less information was transmitted with a reduction of KCC2 activity (Doyon et al., 2015). In addition, impaired KCC2 activity can lead to the loss of pathway specificity in LTP, which could also be seen as a defective information transfer (Figure 5C). As such, in aging animals with impaired KCC2, LTP loses its specificity to stimulated synapses and spreads to other, unstimulated synapses (Ferando et al., 2016). This broadcast of synaptic plasticity may underlie cognitive deficits in aging animals.

Cl− Dysregulation as a Hub of Pathological Dysfunctions

Finally, abnormal sensory processing is another critical consequence of disinhibition by altered Cl gradients in the spinal cord. KCC2 hypofunction is both necessary and sufficient to explain the decrease in threshold of spinal nociceptive output neurons (Lavertu, Cote, & De Koninck, 2014). Loss of KCC2 also unmasks interconnections in spinal sensory circuits, which can explain tactile allodynia (Figure 5D; Keller et al., 2007; Lavertu et al., 2014). Here too, KCC2 blockade can replicate the occurrence of spontaneous bursts of activity in spinal projection neurons after nerve injury, which can explain spontaneous pain events, a hallmark of neuropathic pain (Keller et al., 2007). These abnormal types of excitability/signal processing result from intercellular signaling events that regulate neuronal KCC2. For example, BDNF can be released from nociceptive primary afferents in the dorsal horn after bursting patterns of activity and may contribute to the development of inflammatory pain. On the other hand, BDNF may be released from spinal microglia, after peripheral nerve injury, and cause downregulation of neuronal KCC2 in a TrkB-dependent manner (Coull et al., 2005).This last observation is relevant as it identifies regulation of Cl transport as a mechanism by which immune cells may control synaptic plasticity and neuronal network excitability (Ferrini & De Koninck, 2013).

Conclusion

The Cl gradient, which arises from the distribution of Cl ions inside and outside the cell, critically determines the nature of GABAA/glycine responses. Given the importance of the Cl gradients for the strength of inhibition, the Cl equilibrium is highly regulated in neurons. Indeed, the intracellular Cl is not set at a specific level but is constantly modulated in an activity-dependent manner throughout development and in adulthood. It is clear that the dynamic change in Cl equilibrium has a huge impact on neuronal circuits by affecting the excitability and synaptic plasticity of different neurons. Also, the critical regulation of Cl gradients provides a means by which changes in ECM or the activity of glial cells may have a direct impact on neuronal excitability. Beyond the conventional ions Ca2+, K+, and Na+ that are known to participate in the regulation of excitability, the delicate balance in Cl homeostasis has arisen as an additional pivotal ionic mechanism that regulates neuronal excitability. The balance in Cl gradients can be also easily overwhelmed in pathological conditions, leading to a complete collapse of inhibition. Therefore, the maintenance of Cl gradients may be the common thread that underlies periods of enhanced plasticity, including maladaptive plasticity in disease.

Acknowledgments

We would like to thank Mr. Sylvain Côté for his excellent assistance with the artwork. The work was supported by a Canadian Institutes of Health Research Foundation grants to Y.D.K., a Consejo Nacional de Ciencia y Tecnología scholarship (CONACYT 312229) to J.P.S., and a Canada Research Chair in Chronic Pain and Related Brain Disorders to Y.D.K.

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