Immediately after an ischemic event, large amounts of glutamate contribute to a strong activation of NMDARs that downregulates the expression of both GABA_A and GABA_B receptors through a phosphorylation process activated by high levels of Ca²⁺ (Figure 1) ^{[32][43][44][45][46]}[32,43,44,45,46]. Accordingly, phasic GABA signaling is reduced in the first weeks after stroke ^[28][40][28,40]. This situation further increases neuronal depolarization and subsequent cellular damage. Recently, two proteomic studies have revealed increased levels of the GABA aminotransferase GABT, as well as reduced levels of GABA receptors and the excitatory amino acid transporter EAA2, in the infarct core area from postmortem tissue samples of stroke patients ^[47][48][47,48]. These results validate results from animal models by showing overall decreased GABAergic signaling (elevated catabolism of GABA and reduced GABA receptors) and increased glutamatergic signaling (reduced removal from synaptic cleft by EAA2). That is why the enhancement of GABA signaling at this point can exert a neuroprotective role by decreasing the cellular excitability (Figure 1). Indeed, an early study by Costa and coworkers [49] revealed that the coactivation of both GABA_A and GABA_B receptors promoted neuroprotection in an in vitro model of ischemic stroke. Similarly, the activation of either GABA_A or GABA_B receptors separately also has pro-survival outcomes. Several studies have reported that the remaining GABA_B receptors can be activated between days 1–3 post stroke and this promotes neuroprotection ^[42][50][42,50]. Since the 1990′s, the neuroprotective role of enhancing phasic GABA signaling at the acute phase has been studied throughout pharmacological treatments in both in vitro and in vivo models ^[39][51][39,51]. Likewise, some studies suggest the benefits of enhancing phasic GABA signaling during the chronic phase of stroke in humans ^[52][53][52,53]. It has been reported that the phasic GABA signaling is increased in cortical pyramidal neurons during the chronic phase of stroke [29]. Pharmacological boost of α1 subunit-mediated currents at 3 days post stroke promotes functional recovery by targeting cortical plasticity [29].

Although glutamate is excitotoxic during the acute phase following stroke, it plays a beneficial role during the recovery phase by inducing LTP ^[33][34][33,34]. Indeed, studies in humans have suggested that the stimulation of the penumbra cortex by boosting local excitability as soon as 7 days post stroke improves functional outcome [54]. However, there is an increase in extrasynaptic levels of GABA due to the reduction in the amount of astrogial GABA transporters on day 7 post stroke in mice [28]. This event hyperpolarizes neurons at the penumbra area and negatively modulates the induction of LTP ^[32][34][55][32,34,55]. Indeed, a recent study using magnetic resonance spectroscopy showed that patients with a low excitatory–inhibitory ratio post stroke had a worse motor outcome [56]. The application of pharmacological treatments negatively targeting either all α subunits or only α5 subunit-mediated tonic GABA currents at 3 days post stroke has shown significant behavioral recovery in mouse models ^[28][34][37][28,34,37]. Interestingly, it has been reported that there is a possible role of extrasynaptic GABA_C receptors, a well-known subclass of GABA_A receptors, in these increased tonic currents during post-acute and chronic phases. The application of antagonists targeting GABA_C receptors from day 3 post stroke improved the motor function of injured mice [38]. These results together suggest that the time window for the administration of an extrasynaptic GABA_A receptor blocker without affecting its initial neuroprotective role is around 3 days after the infarct, at least in mice.

Overall, the potentiation of symmetric signaling immediately after ischemic stroke counteracts the prominent excitatory cellular state promoting neuronal survival. Based on murine models, the acute phase lasts 3 days, and one of the most important questions to be solved is the exact duration of this phase in humans in order to replicate the results from animal models to patients. In contrast, during the postacute and chronic phase, the rise in tonic GABAergic signaling has to be blocked in order to achieve a better functional outcome. Curiously, the potentiation of phasic inhibition is beneficial during the recovery state. It would be interesting to combine pro-GABA drugs during the acute phase and then change them progressively to both prophasic signaling and antitonic currents.

2.2. Cation–Chloride Cotransporters

Ionotropic GABAergic signaling is primarily supported by the chloride ion gradient across the plasmatic membrane [57]. In mature neurons, the regular GABA_A-mediated transmission leads to hyperpolarization by allowing the entry of Cl⁻ ions that increases the intracellular concentration of chloride ([Cl⁻]_i) (Figure 2) ^[26][57][26,57]. The maintenance of [Cl⁻]_i mainly depends on cation–chloride cotransporters, where Na⁺-K⁺-2Cl⁻ cotransporters (NKCCs) and K⁺-Cl⁻ cotransporters (KCCs) are the most important in the CNS ^[26][58][26,58]. The isoform NKCC1 is the only one expressed in the CNS, and its function is to increase the [Cl⁻]_i. In contrast, the isoform KCC2 is the main one responsible for decreasing [Cl⁻]_i in mature neurons ^[26][58][26,58].

Figure 2. Role of NKCC1 and KCC2 cotransporters in the effect of Cl⁻ flow through GABA_A receptor. (Health). In a normal situation, the expression of both cotransporters leads to a higher [Cl⁻]_e compared to the [Cl⁻]_i. This results in neuronal hyperpolarization when GABAA receptors are activated; (Injury) After damage, there is an osmotic stress that activates the WNK pathway. This ultimately ends in the phosphorylation of both cotransporters with different outcomes, the expression of KCC2 decreases, whereas the expression of NKCC1 increases; leading to a higher [Cl⁻]_i compared to the [Cl⁻]_e, which results in depolarizing GABA_A receptor-mediated responses.

It has been well-documented that under some pathological conditions, [Cl⁻]_i can be dysregulated, leading to a depolarizating effect mediated by GABA_A receptors (Figure 2) [59]. This is primarily motivated by a high expression of NKCC1 and a low expression of KCC2, leading to a higher [Cl⁻]_i compared to the extracellular concentration of chloride ([Cl⁻]_e) ^[57][58][60][57,58,60]. The main cellular cascade involved in the regulation of NKCC1 and KCC2 following osmotic stress (low [Cl⁻]_i) is the With-No-Lysine (K) (WNK) pathway, which ultimately phosphorylates both NKCC1 and KCC2 with opposite outcomes: NKCC1 is activated whereas KCC2 is inhibited (Figure 2) ^[61][62][61,62].

There is compelling evidence showing an increase in neuronal NKCC1 expression immediately after ischemic stroke, an event that contributes to cellular hyperexcitability and cell death ^{[45][60][63][64][65][66]}[45,60,63,64,65,66]. Wang and colleagues [60] have shown that NKCC1 is significantly upregulated in cortical neurons from 3 h to 48 h following focal cerebral ischemia in a rat model. Acute pharmacological treatment using bumetanide, an NKCC1 inhibitor drug, revealed a neuroprotective effect by increasing neuronal survival in an in vitro model of stroke [45]. This is concordance with previous in vivo results showing a reduction in the infarction volume as well as in the ischemic necrotic cell death, especially remarkable when bumetanide was applied preinjury ^{[60][63][67][68]}[60,63,67,68]. A recent study has shown that the inhibition of NKCC1 from day 7 post stroke enhanced axonal sprouting from uninjured neurons, resulting in a significant behavioral improvement [66].

As previously shown, stroke triggers the WNK signaling pathway leading to both the activation of NKCC1 and the inhibition of KCC2 via phosphorylation (Figure 2) ^[62][69][62,69]. Indeed, the activation of the WNK signaling pathway significantly increased the activity of NKCC1 in cortical and striatal neurons at 6 and 24 h after ischemic stroke in mice [65]. These findings suggest that blocking the activation of the WNK cascade offers a new therapeutic target to improve the outcome following stroke by targeting NKCC1 activation ^{[62][69][70][71][72]}[62,69,70,71,72].

Contrary to what it was seen for NKCC1, the amount of KCC2 at both mRNA and protein levels was downregulated in both rat and mouse models of ischemic stroke ^{[45][64][66][73][74]}[45,64,66,73,74]. Curiously, whereas the KCC2 levels in the plasma membrane are notably reduced 3 h post ischemia [74], there is a progressive decrease in the levels of total KCC2 given that it is significant on days 1 and 7 post stroke, but not at 2 h after injury [64]. Therefore, a relationship between a maintained expression of KCC2 overtime and the long-term survival rate of neurons has been proposed [64], which was recently supported ^[26][74][26,74]. In this study, hippocampal pyramidal neurons had regular levels of KCC2 and did not display damage signals at 6 h post stroke, but they started to degenerate when KCC2 levels decreased at 48 h after stroke [26]. In a similar way, an acute blockage of upstream pathways inhibiting KCC2 showed an increased neuronal survival following an ischemic incident in mice [74]. Therefore, all these evidences seem to point at the upregulation of KCC2 as a therapeutic target to provide protection against stroke-induced cell death. However, similar to the manipulation of GABA signaling, it is a challenge to decipher the timing between acute and recovery phase in humans, and hence, to find the correct point at which to change KCC2 expression/activity from increased to decreased in order to achieve further functional outcome after ischemic stroke [26].

In summary, blocking NKCC1 during the first hours post stroke has a remarkable effect on neuronal outcome by reducing necrotic death. Likewise, increasing KCC2 levels displays a beneficial role at least during the acute phase, which raises the interesting question of what would happen if KCC2 was manipulated during the post-acute/chronic phase, since higher levels of KCC2 would induce GABA-mediated hyperpolarization leading to a tonic currents-like effect.