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Keywords:

  • GABA receptor;
  • KCC2;
  • NKCC1;
  • Chloride cotransporter;
  • Kainic acid;
  • Neonatal;
  • Adult;
  • Hippocampus;
  • Rat;
  • Epilepsy

Abstract

  1. Top of page
  2. Abstract
  3. CHLORIDE HOMEOSTASIS AND GABAA RECEPTOR SIGNALING
  4. PATHOLOGICAL REEMERGENCE OF DEPOLARIZING GABAAERGIC RESPONSES
  5. EFFECTS OF NEONATAL SEIZURES ON CHLORIDE HOMEOSTASIS
  6. CONCLUDING REMARKS
  7. Acknowledgments
  8. REFERENCES

Summary:  GABAA receptors have dual functions during development. They depolarize immature neurons but hyperpolarize more mature neurons. This functional switch has been attributed to age-related differences in the relative abundance of cation chloride cotransporters, such as KCC2 and NKCC1, which regulate chloride homeostasis. Certain insults, such as trauma, ischemia, and seizures, if they occur when GABAAergic signaling is hyperpolarizing, such as in the adult brain, can lead to reappearance of the immature, depolarizing synaptic responses to GABAA receptor activation. In certain cases, this has been associated with either reduced expression of KCC2 or increase in NKCC1. In epilepsy, the depolarizing effects of GABAA receptors have been proposed to be important for the acquisition and/or maintenance of the epileptic state. Using the kainic acid model of status epilepticus, we have studied the effects of repetitive neonatal episodes of status epilepticus on the expression of cation chloride cotransporter KCC2 in the neonatal hippocampus. In contrast to adults, seizures increased KCC2 mRNA expression in the CA3 region of the neonatal hippocampus. The contrasting patterns of regulation of KCC2 by seizures in mature and immature neurons may be one of the age-related factors that protect the neonatal brain against the development of epilepsy.

GABAA receptors have multifaceted actions upon the function and differentiation of the brain. Apart from their complex pharmacology and network effects, attributed to the large number of subunit combinations, receptor modulators, and subcellular sites of action, their ability to either depolarize or hyperpolarize neurons further amplifies the degrees of complexity (De Groat, 1972; Brown and Scholfield, 1979; Mueller et al., 1983; Cherubini and North, 1984; Ben-Ari et al., 1989; Barnard et al., 1998; Brooks-Kayal et al., 2001). The switch of GABAA receptors, from being depolarizing early in life to its classical hyperpolarizing effects later on, occurs at different time points, in different regions (Mueller et al., 1983; Rivera et al., 1999). GABAA receptor-mediated depolarizations can activate voltage-sensitive calcium channels and initiate a cascade of calcium sensitive signaling processes important for neuronal differentiation and survival (Reichling et al., 1994; Staley et al., 1995; Owens et al., 1996; Ben-Ari, 2002). In the pyramidal neurons of the rat hippocampus, the switch occurs by the end of the second postnatal week (Rivera et al., 1999; Khazipov et al., 2004). In certain regions, such as the substantia nigra, the timing of GABAA receptor switch is different between males and females (Galanopoulou et al., 2003; Kyrozis et al., 2006) and this is important for the sexual differentiation of these brain structures (Galanopoulou, 2005).

CHLORIDE HOMEOSTASIS AND GABAA RECEPTOR SIGNALING

  1. Top of page
  2. Abstract
  3. CHLORIDE HOMEOSTASIS AND GABAA RECEPTOR SIGNALING
  4. PATHOLOGICAL REEMERGENCE OF DEPOLARIZING GABAAERGIC RESPONSES
  5. EFFECTS OF NEONATAL SEIZURES ON CHLORIDE HOMEOSTASIS
  6. CONCLUDING REMARKS
  7. Acknowledgments
  8. REFERENCES

The decision as to whether GABAA receptor activation will depolarize or hyperpolarize a neuron is set by factors that control the gradients of anions that flow through open GABAA receptors, namely Cl- and HCO3-. Chloride homeostasis is controlled mostly by cation chloride cotransporters. Principal representative of Cl- extruding cotransporters in neuronal cells is KCC2 (Payne et al., 1996), whereas the most studied importer of Cl- is NKCC1 (Moore-Hoon and Turner, 1998). The developmental increase in KCC2, which eventually overcomes the influence of NKCC1, is required for the appearance of the mature, hyperpolarizing synaptic GABAAergic responses in hippocampal neurons (Rivera et al., 1999; Hubner et al., 2001). Another controller is carbonic anhydrase VII (Car7 or CA VII), the expression of which has been shown to increase in hippocampus by postnatal day 13 (PN13). This coincides with the time when the high-frequency stimulation-induced tonic GABAAergic excitation appears (Ruusuvuori et al., 2004).

Apart from the developmental factors, which control the direction of the synaptic or tonic effects of GABAA receptor activation, a major focus of research has been thrust around the way these processes are aberrantly regulated in pathologic conditions. Emerging evidence, stemming mostly from studies in mature neurons, indicate that under certain pathological conditions, reappearance of the depolarizing GABAAergic responses occurs. In some cases, as in traumatic injuries, this can be considered an attempt to reequip the mature neurons with the regenerating properties of the immature neurons and aid in the healing of the insulted brain. In epilepsy, emerging evidence suggests that this functional regression of GABAA receptors is a critical feature of the epileptic state. This manuscript will review these findings and discuss them in view of our first data on the effects of neonatal seizures on the factors that control the function of the GABAA receptors.

PATHOLOGICAL REEMERGENCE OF DEPOLARIZING GABAAERGIC RESPONSES

  1. Top of page
  2. Abstract
  3. CHLORIDE HOMEOSTASIS AND GABAA RECEPTOR SIGNALING
  4. PATHOLOGICAL REEMERGENCE OF DEPOLARIZING GABAAERGIC RESPONSES
  5. EFFECTS OF NEONATAL SEIZURES ON CHLORIDE HOMEOSTASIS
  6. CONCLUDING REMARKS
  7. Acknowledgments
  8. REFERENCES

Both in vivo and in vitro experiments have shown that a variety of insults, when targeting neurons with hyperpolarizing GABAAergic responses, lead to the reappearance of depolarizing GABA responses with ensuing calcium transients. Such insults include trauma (Topolnik et al., 2003; Bonislawski et al., 2006), high temperature, hypotonic or hypertonic environment (van den Pol et al., 1996), nerve transections (van den Pol et al., 1996; Coull et al., 2003; Toyoda et al., 2003), oxygen-glucose deprivation (Galeffi et al., 2004; Pond et al., 2004). This reversal of GABAA receptor function is often attributed to a decrease in KCC2 mRNA (Coull et al., 2003; Toyoda et al., 2003; Galeffi et al., 2004; Bonislawski et al., 2006), whereas NKCC1 does not necessarily change (Galeffi et al., 2004). The reappraisal of the excitatory GABAAergic signaling has been interpreted as a regenerative process, aiming to stimulate calcium regulated gene expression or promote neuronal differentiation or survival (Franklin and Johnson, 1992; Toyoda et al., 2003). However, it can also be the instigator of acute or long-term adverse sequelae. In peripheral nerve injuries, KCC2 downregulation and the ensuing depolarizing GABAAergic signaling reduce the threshold for neuropathic pain (Coull et al., 2003). In the oxygen-glucose deprivation model, they contribute to anoxia-induced death (Galeffi et al., 2004; Pond et al., 2004).

Of particular relevance to the epilepsy field are recent findings from the subiculum of human epileptic tissue, which describe depolarizing GABA responses in the pyramidal neurons of the network that generates interictal-like activity (Cohen et al., 2002). Similar to the previous reports by Kohling et al. (Kohling et al., 1998), in human neocortical temporal lobe tissue, these interictal discharges were blocked by GABAA receptor antagonists. Palma et al. showed that the excitatory effects of GABAA receptors in cell membranes obtained from temporal lobectomies of epilepsy patients were attributed to increased NKCC1 and decreased KCC2 expression (Palma et al., 2006).

Animal studies have also linked the depolarizing GABAAergic signaling with the epileptic state. In a model of postlesional neocortical epileptogenesis, using “undercut and transcortical” cortical transections, impaired Cl- extrusion in layer V pyramidal neurons was noted at the time when ictal or interictal epileptiform patterns were expected to occur (Hoffman et al., 1994; Jin et al., 2005). No significant difference was observed between lesioned and intact cortex in regards to the reversal potential for chloride (ECl). However, the lesioned cortex showed more positive shifts in ECl under conditions of chloride loading, as are expected to occur under high-frequency repetitive stimulation, often seen in seizures. The authors suggest that this may be a mechanism underlying the increased excitability of lesioned cortex. Kindling of adult rodents has also been reported to alter the balance of cation chloride cotransporters and favor the appearance of depolarizing GABA responses. Hippocampal kindling acutely decreased KCC2 expression in the hippocampus, through activation of the brain derived neurotrophic factor (BDNF) and tyrosine kinase receptor B (TrkB) signaling pathway (Rivera et al., 2002). On the other hand, amygdala kindling of adult rats, increased NKCC1, decreased KCC1 and ClC-2 in the dentate gyrus, but did not alter KCC2 (Okabe et al., 2003). These changes again favor the maintenance of high intracellular chloride concentrations and excitatory GABAAergic signaling.

These studies convincingly suggest that the reappraisal of the excitatory GABAA receptor signaling in the epileptic brain may, at least in some occasions, be implicated in the generation of interictal discharges. A common clinical experience is that interictal discharges do not respond to the current antiepileptic drugs, even when seizures do. If ictal events have different origin than interictal events, what is the role of excitatory GABAAergic signaling in ictogenesis? In the studies of Kohling et al. in human epileptic neocortical temporal lobe tissue, only the interictal sharp waves, but not the seizure events, were blocked by the GABAA receptor blocker bicuculline (Kohling et al., 1998). Compelling evidence, however, that the seizure-induced reappearance of depolarizing GABAA receptor responses may in certain cases be also important for ictogenesis was presented by Khalilov et al (Khalilov et al., 2003). The authors studied intact hippocampal formations from PN6-7 Wistar male rats, using the triple chamber technology (Khalilov et al., 2003). In this preparation, hippocampal pyramidal neurons already exhibited hyperpolarizing GABAAergic postsynaptic responses. Commissural propagation of kainate-induced seizures from one hippocampus to the contralateral drug-naïve hippocampus increased ECl and rendered the hippocampus capable of generating spontaneous seizures driven by these excitatory effects of GABAA receptors (Khalilov et al., 2003). In a continuation of these studies, the authors reported that propagation of fast oscillations, which depend on GABAAergic depolarizations, was required for the generation of spontaneous ictal events from the drug-naive immature hippocampus (Khalilov et al., 2005). Interestingly, functional GABAergic synapses were not a requirement for adult hippocampi to manifest spontaneous seizures (Khalilov et al., 2005). This seems to agree with the observations that GABAAergic blockade failed to suppress ictal events in adult epileptic preparations, albeit GABAA receptors were depolarizing (Kohling et al., 1998). These data emphasize the differences between immature and adult brain in regards to susceptibility to, generation of, and the impact of seizures.

EFFECTS OF NEONATAL SEIZURES ON CHLORIDE HOMEOSTASIS

  1. Top of page
  2. Abstract
  3. CHLORIDE HOMEOSTASIS AND GABAA RECEPTOR SIGNALING
  4. PATHOLOGICAL REEMERGENCE OF DEPOLARIZING GABAAERGIC RESPONSES
  5. EFFECTS OF NEONATAL SEIZURES ON CHLORIDE HOMEOSTASIS
  6. CONCLUDING REMARKS
  7. Acknowledgments
  8. REFERENCES

How applicable can these observations be in the case of immature rats, prior to the timing of GABAA receptor switch? We have recently shown that the regulation of KCC2 expression differs in neurons with depolarizing versus hyperpolarizing GABAA receptor responses (Galanopoulou and Moshe, 2003; Galanopoulou et al., 2003). In neurons that are depolarized by GABAA receptor agonists, like muscimol, KCC2 mRNA is upregulated by muscimol and downregulated by GABAA receptor blockers, like bicuculline. In contrast, in neurons with hyperpolarizing GABAAergic responses, muscimol decreases KCC2 mRNA. Since during seizures there is increased activation of GABAA receptors, we hypothesized that the transcriptional regulation of KCC2 by seizures may also be different depending on the functional status of GABAA receptors.

To address this question, male Sprague-Dawley pups were subjected to three episodes of status epilepticus (SE) by intraperitoneal (ip) injections of kainic acid (KA), once daily at PN4, PN5, and PN6. During this developmental period, the CA3 pyramidal cells of the hippocampus still exhibit depolarizing GABAA receptor responses (Khazipov et al., 2004, and our own preliminary data). Using these doses, all rats developed SE with persisting behavioral seizures for at least 6 h of observation, and minimal mortality. Seizure behavior consisted of scratching, hindlimb clonus, and tonic seizures alternating with swimming-like behavior. Controls were kept also separate from the dam for the same period. Following each observation period, pups were returned to their dam. Behaviorally, rats seemed similar to controls the day after the SE. One set of pups was anesthetized, and transcardially perfused at PN7, and brains were stained with Fluoro-Jade B to identify neurodegenerating neurons (protocol as per manufacturer's instructions, CHEMICON, Temecula, CA, U.S.A.) (Schmued et al., 1997). A second set of pups was euthanized at PN10 and brains were fast frozen and processed for KCC2-specific digoxigenin-labeled in situ hybridization, as previously described (Galanopoulou et al., 2003). In all experiments, controls were assayed concurrently with the rats, which were subjected to SE. Cellular expression of KCC2 mRNA over the CA3 pyramidal layer of the anterior dorsal hippocampus was compared with densitometry using the Scion Image 1.63 software (NIH, Bethesda, MD, U.S.A.), as described previously (Galanopoulou et al., 2003), with the experimenter blinded to the identity of the sections.

At PN7, there was no significant injury in the hippocampus of rats subjected to 3 episodes of KA-SE, as compared to the control rats (Fig. 1, n = 5 per group). At PN10, KCC2 mRNA was significantly increased in the CA3 region of the rats subjected to KA-SE at PN4-6. KCC2 mRNA signal intensity was 88% greater than the controls (Fig. 1, panel B, n = 5 per group). These findings contrast with the patterns of KCC2 regulation by seizures in older animals. Although model specific differences cannot be excluded yet, they also strongly agree with the initial hypothesis that the translational effects of SE in animals with still depolarizing GABAA receptor responses is different than those observed in animals with hyperpolarizing GABAA responses. Other age-related differences in signaling pathways, whether related or unrelated to GABA signaling, may be involved. For instance, kainic acid seizures are known to release BDNF in the CA3 region, both in neonatal pups (Kornblum et al., 1997) and in adults (Rudge et al., 1998). However, BDNF has age-dependent effects on KCC2 expression in hippocampus. BDNF increases KCC2 in very immature neurons (Aguado et al., 2003), but decreases it in the more mature (Rivera et al., 2002). The differential regulatory effects of BDNF on KCC2 in young versus older neurons could be due to the utilization of different signaling pathways, as has recently been proposed by Rivera et al. (Rivera et al., 2004).

image

Figure 1. Three episodes of KA-SE at PN4-6 do not produce significant injury in male CA3 pyramidal layer at PN7, but increase KCC2 mRNA at PN10. A: Fluoro-Jade B staining (200× magnification) of an adult male rat subjected to a single episode of lithium-pilocarpine SE shows significant injury in the curve of the CA3 (CA3a region), 48 h after the SE. Fluoro-Jade B stained cells are green, as shown by the white arrowheads (left panel). In contrast, no significant injury is observed in the same region of a PN7 male rat, subjected to 3 episodes of KA-SE, each at PN4, PN5, and PN6 (right panel). The white arrows show the lateral border of the curve of the CA3 region. B: Male PN10 rats with a prior history of KA-SE at PN4-6 demonstrated stronger cellular densitometric signal for KCC2 mRNA in the CA3a region of the hippocampus (shown by the rectangular), compared to controls. The panel presents representative photographs in a 40× magnification.

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CONCLUDING REMARKS

  1. Top of page
  2. Abstract
  3. CHLORIDE HOMEOSTASIS AND GABAA RECEPTOR SIGNALING
  4. PATHOLOGICAL REEMERGENCE OF DEPOLARIZING GABAAERGIC RESPONSES
  5. EFFECTS OF NEONATAL SEIZURES ON CHLORIDE HOMEOSTASIS
  6. CONCLUDING REMARKS
  7. Acknowledgments
  8. REFERENCES

These studies show the plasticity of GABAA receptors and their regulatory factors in response to stressful conditions, such as seizures, trauma, or ischemia. In adult neurons, the functional regression of the GABAAergic signaling may act as a healing mechanism but may also contribute to adverse pathological outcomes. In the case of epilepsy, the reappearance of depolarizing GABAAergic responses in adult neurons may add to the excitability of the brain. However, there is no evidence yet that it is sufficient to create spontaneous seizures. In developing neurons in which GABAAergic signaling is already hyperpolarizing, there is in vitro evidence that prolonged seizures can trigger the reappearance of depolarizing GABAAergic responses, which can be ictogenic.

In contrast, the observed KA-SE-induced increase in KCC2 expression in the neonatal hippocampus with still depolarizing GABAAergic signaling would not favor the maintenance of excitatory GABAAergic effects. As depolarizing GABAA receptors have been shown to be ictogenic at this age (Khalilov et al., 2003; Dzhala et al., 2005; Khalilov et al., 2005), this could be a protective, balancing mechanism to diminish the propensity of the already highly vulnerable neonatal brain to manifest seizures, whether evoked or self-generated. It could therefore be one of the reasons why the neonatal brain, albeit highly susceptible to seizures, is more resistant to the development of epileptogenesis.

However, if there is one humbling lesson to learn from GABA research is that GABAA receptor physiology changes not only during development but also by mutual, dynamic interactions with the local environment. It is therefore important to study how the observed changes will impact on GABAA receptor physiology, in the context of a hippocampus potentially altered by the prior seizures, both at rest and under a stressful situation, such as seizures. Moreover, if the observed changes in KCC2 do influence the timing of GABAA receptor switch, it would be important to define how this would impact on the differentiation and function of the hippocampus, as it is known that prolonged or repetitive neonatal seizures may have long-term cognitive effects (Sogawa et al., 2001).

Acknowledgments

  1. Top of page
  2. Abstract
  3. CHLORIDE HOMEOSTASIS AND GABAA RECEPTOR SIGNALING
  4. PATHOLOGICAL REEMERGENCE OF DEPOLARIZING GABAAERGIC RESPONSES
  5. EFFECTS OF NEONATAL SEIZURES ON CHLORIDE HOMEOSTASIS
  6. CONCLUDING REMARKS
  7. Acknowledgments
  8. REFERENCES

Acknowledgments:  I wish to thank Dr. Solomon L. Moshé for valuable feedback on this manuscript and Mrs. Qianyun Li for excellent technical assistance. This study was supported by NINDS grants NS45243 and NS20253 and a grant from the Rett Syndrome Research Foundation.

REFERENCES

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  2. Abstract
  3. CHLORIDE HOMEOSTASIS AND GABAA RECEPTOR SIGNALING
  4. PATHOLOGICAL REEMERGENCE OF DEPOLARIZING GABAAERGIC RESPONSES
  5. EFFECTS OF NEONATAL SEIZURES ON CHLORIDE HOMEOSTASIS
  6. CONCLUDING REMARKS
  7. Acknowledgments
  8. REFERENCES
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