Epilepsia, 46(Suppl. 7):29–38, 2005 Blackwell Publishing, Inc. C International League Against Epilepsy
Rearranging Receptors Amy R. Brooks-Kayal Children’s Hospital of Philadelphia, University of Pennsylvania, Abramson Research Center, Philadelphia, Pennsylvania, U.S.A.
Summary: The immature brain is highly susceptible to seizures. The heightened susceptibility to seizures appears to be due, at least in part, to developmental changes that skew the balance between excitatory and inhibitory neurotransmitter systems in the brain in favor of a state of excitation. Multiple factors, including changes in GABAergic and glutaminergic receptor composition, number, and distribution, all contribute to produce the characteristic limbic hyperexcitability seen during the early postnatal period. Infants and young children who experience prolonged or repetitive seizures have
an increased risk of subsequently developing epilepsy. Evidence to date suggests that status epilepticus produces permanent changes in the molecular and cellular structure of limbic circuitry that, in turn, result in a long-lasting increase in hippocampal excitability and lower seizure thresholds in later life. Key Words: Epilepsy—Development—Neonatal seizure—Early-life seizures—Glutaminergic—GABAergic— Seizure thresholds—Mirror focus—Receptor development— Subunit expression—Neurotransmitters—GABA—Glutamate.
Epidemiologic studies clearly show that the highest incidence of seizures occurs during the first decade of life in humans, particularly during the first year of life (1,2). In addition, animal studies have demonstrated that there is enhanced susceptibility to experimentally induced seizures of all types during the early postnatal period (3,4). Cumulative evidence suggests that the hyperexcitability of the immature brain is mediated by developmental changes in the structure and function of limbic circuitry. The following section will briefly review GABAergic and glutaminergic receptor physiology, highlight the most significant changes observed in these neurotransmitter systems during the early postnatal period, and discuss their potential impact on seizure susceptibility in the developing brain.
maturation of excitatory systems on the other. Gammaaminobutyric acid (GABA) is the main inhibitory neurotransmitter in the adult brain. Epileptologists have been interested in this system because commonly prescribed antiepileptic drugs (AEDs), such as phenobarbital, the benzodiazepines, and to a lesser extent valproate, topiramate, and levitiracetam, reduce seizure activity by augmenting GABA receptor activity. The GABAergic system consists of three main receptor subtypes: GABAA , GABAB , and GABAC . GABAA receptors are primarily located postsynaptically and mediate most of the fast synaptic inhibition in the brain. They are anion selective and gate primarily chloride, although under certain circumstances they may also gate bicarbonate. GABAA receptors are heterogeneous complexes composed of multiple protein subunits. Numerous subtypes exist for each subunit. The wide variety of combinations of subunits and their subtypes confers a broad range of functional and pharmacological properties to the receptor. The γ subunit, for example, is required for GABAA receptors to be responsive to benzodiazepine-type drugs, whereas the α subunit subtype determines the type of the benzodiazepine binding site (e.g., I or II) (5,6). Brain regions that express the highest concentration of the α1 subunit have a correspondingly high number of type 1 benzodiazepine binding sites and are, in turn, more sensitive to zolpideminduced augmentation and less sensitive to zinc-induced inhibition (7–9). GABAB receptors are G-protein-linked metabotropic receptors that are located both presynaptically and postsynaptically and are responsible for the slower, more
WHY IS THE IMMATURE BRAIN MORE SUSCEPTIBLE TO SEIZURES? The development of GABA inhibitory systems Early in the development, there is an imbalance between excitation and inhibition in the brain characterized by the relatively slow maturation of inhibitory neurotransmitter systems on the one hand and the rapid, exuberant Address correspondence and reprint requests to Amy R. Brooks-Kayal at Children’s Hospital of Philadelphia, University of Pennsylvania, Abrahamson Research Center, 3615 Civic Center Blvd, Room 502, Philadelphia, PA 19104-4318, U.S.A. E-mail:
[email protected] This supplement is cosponsored by the American Epilepsy Society and the Center for Advanced Medical Education, Inc. Support for this activity has been made possible through an educational grant from Pfizer Inc.
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FIG. 1. This schematic illustrates how the developmental shift in the chloride gradient (Cl− ) affects GABAergic transmission in the immature and mature brain. GABAA R, GABAA receptor; KCC2, transports Cl− out of the cell; NKCC1, transports Cl− into the cell; mRNA, messenger RNA; Vm , membrane voltage; ECl , chloride equilibrium potential [from Rivera et al., 1999 (13); Plotkin et al., 1997 (14); Clayton et al., 1998 (15); Lu et al., 1999 (16); Ganguly et al., 2000 (17)].
long-lasting inhibitory currents. Like GABAA receptors, they are composed of multiple subunits, primarily R1 and R2, which have additional diversity due to splice variation. Also like GABAA , GABAB receptors are widely distributed in the central nervous system (CNS), particularly in the hippocampus, cerebellum, and thalamus. In contrast, GABAC receptors are located primarily in the retina and do not appear to play a significant role in epilepsy. The function of the GABAergic system differs markedly in the mature and immature brain. Whereas GABAA receptor activation results in neuronal hyperpolarization and an inhibition of cell firing in the mature brain, receptor activation results in membrane depolarization and excitation in the immature brain (10–12). The switch from GABA-mediated excitation to inhibition appears to be related to changes in the chloride gradient that occur during the course of development (13–18) (see Fig. 1). In mature neurons, the intracellular concentration of chloride is low due to the presence of KCC2, a chloride extruding transporter. When GABAA receptors are activated, chloride flows along the concentration gradient into the cell, causing hyperpolarization and hence inhibition of the postsynaptic response. In contrast, intracellular concentrations of chloride are high in immature brain due to the combined effects of low KCC2 expression and the presence of NKCC1 transporters that actively carry chloride into the neuron. When GABAA receptors are activated, ion channels open, chloride flows out of the cell, and depolarization occurs. In rodents, KCC2 expression is very low during the first two postnatal weeks. By inference it is thought that expression is low in humans until around the end of gestation (19). Epilepsia, Vol. 46, Suppl. 7, 2005
A number of laboratories have shown that depolarizing (e.g., excitatory) GABA currents are critical for the development of calcium-dependent processes, such as neuronal proliferation, migration, targeting, and synaptogenesis (20–24). In addition, there is evidence suggesting that GABA currents also play a critical role in the generation of ictal activity in the developing brain. It has been known for some time that synchronous neuronal activity in the hippocampus can be driven by GABAA receptor activation and inhibited by GABAA receptor blockade (25). More recent evidence, however, suggests that GABA-mediated excitation may drive ictal activity in the developing hippocampus as well (26,27). When Dzhala and Staley (26) looked at the generation and propagation of seizure activity in cultured hippocampal slices from juvenile rats, they found that the ictal-like epileptiform activity induced by high extracellular potassium levels was exacerbated by the GABAA receptor agonist, muscinol, and inhibited by the antagonist, bicuculline. This is the opposite of what is typically observed in the hippocampus of adult rats. Neurologists are always concerned that a secondary epileptogenic mirror focus may develop in a child who is having repetitive seizures. In a recent series of experiments, Khalilov and colleagues demonstrated the formation of a mirror focus in immature rat hippocampi and identified GABA-mediated excitation as one of the mechanisms underlying its induction (27,28). The two hippocampi and connecting commissural fibers were harvested from 7-day-old rats and placed in individual chambers that could be independently perfused. When the hippocampal commissural fibers were intact, the application of a single dose of kainic acid to one side of
REARRANGING RECEPTORS the hippocampus induced synchronized ictal activity that immediately spread to the opposite side and then subsided. No subsequent spontaneous epileptiform activity was observed. However, when kainic acid was administered repeatedly to the ipsilateral side and then the connections between the two hippocampi were pharmacologically blocked by the application of the sodium channel blocker, tetrodotoxin (TTX), spontaneously generated ictal discharges were recorded from the contralateral side. The fact that the GABAA antagonist, bicuculline, blocked the seizures in the secondary focus suggests that the excitatory actions of GABA were critical to the generation of the mirror focus. Data from a variety of laboratories indicate that there are also clear regional and developmental differences in GABAA receptor subunit expression. Whereas the expression of certain subunits, such as α2, α3, and α5, peak early in development and then stabilize or decline, the expression of others, such as α1 and γ 2, are low at birth and then progressively increase to adult levels (29,30). These developmental changes in subunit expression are associated with a shift in GABAA receptor physiology toward more rapid kinetics, increased sensitivity to augmentation by the type 1 benzodiazepine agonist, zolpidem, and decreased sensitivity to the inhibitory effects of zinc (31,32). In humans, there is a three-fold increase in the expression of the α1 subunit in both the cortex and cerebellum between 36 weeks gestation and adulthood (33). As shown in Fig. 2, α1 subunit expression is very low during late gestation and the early postnatal period. A closer inspection of subunit development has revealed that very specific changes occur within individual neurons within the lim-
FIG. 2. α1 subunit mRNA expression in human cortex and cerebellum between 36-week gestation and adulthood [from Brooksc 1993 Wiley-Liss, Inc., Kayal and Pritchett, 1993 (33)] Copyright A Wiley Company. Reproduced with permission of John Wiley & Sons, Inc.
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bic system during the early postnatal period. For example, in rats there is more than a two-fold increase in the expression of the α1, β1, and γ 2 subunits in the dentate gyrus between postnatal day 7 (P7) and adulthood and a concomitant shift in receptor physiology similar to that described above. Fig. 3 depicts the developmental changes in GABAA receptor expression and function observed in individual dentate granule neurons of rats on P5 to P7, P17 to P21, and in adulthood (9). It is apparent that there are profound differences in GABAA receptor activity over the course of development. Immature dentate neurons have fewer receptors, lower GABA-mediated currents, are less sensitive to zolpidem augmentation, and are more sensitive to zinc-induced inhibition. The dentate gyrus functions as an inhibitory gate by controlling the flow of information between the entorhinal cortex and the hippocampus. Thus, the clinical ramifications of these findings are far reaching. Infants and young children have a uniquely immature GABA system that is not equipped to handle the most frequently prescribed anticonvulsant drugs, phenobarbital and the benzodiazepines, which act by augmenting GABAA receptor activity. Very early in development, the GABAergic system may be either insensitive to AEDs or respond in a way opposite to what is expected (i.e., augmenting excitation). Later on, as inhibitory systems gradually mature, they may still be hypofunctional and less sensitive to pharmacological manipulation. The success of treating young patients, therefore, will depend on the development of new AEDs with novel mechanisms of action that target the unique functional status of the immature brain. GABAB receptors, which are located both presynaptically and postsynaptically, also undergo distinct physiological and functional changes during early development. In rodents, the presynaptic expression of the GABAB1a subunit predominates at birth and then declines to adult levels by P14 (34). In comparison, the postsynaptic GABAB1b subunit is low at birth, rises during the first two postnatal weeks, and then declines to adult levels (35). From a functional standpoint, therefore, there is some presynaptic GABAB receptor activity at birth, but no postsynaptic function until the second or third postnatal week (36). Hence both GABAB and GABAA inhibition are low during the early postnatal period. The development of glutaminergic excitatory systems The balance between excitation and inhibition in the immature brain is skewed, with the effects of excitatory neurotransmitter systems predominating initially and then becoming less predominant as inhibitory systems gradually mature. Glutamate is the primary excitatory neurotransmitter in the CNS and acts on two major receptor subtypes: ionotropic receptors that are ligandgated, cation-selective channels (e.g., NMDA, AMPA, kainate) and metabotropic receptors that are linked to Epilepsia, Vol. 46, Suppl. 7, 2005
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FIG. 3. Developmental changes in GABAA receptor expression and function observed in individual neurons from the dentate gyrus of rats at three different ages. Upper graph: Total GABAA receptor subunit (GABAR) mRNA expression and the relative expression of each individual subunit. Lower graphs: Mean maximal current evoked by 1 mM GABA, mean percent augmentation of the 10 µM GABA response by 100 nM Zol (zolpidem), and mean percent inhibition by 100 µM zinc [from Brooks-Kayal et al., 2001 (9)]
G-proteins and the regulation of second messengers (37,38). N-methyl-D-aspartate (NMDA) receptors are composed of multiple subunits, and subtypes of each subunit exist, giving them a wide range of functional activities. They are located mainly on postsynaptic neurons and are unique in that they gate both sodium and calcium ions. In addition, NMDA receptors require glycine as a coagonist for activation and have a voltage-dependent magnesium blockade that must be released before agonist binding can open the channel. β-amino-3-hydroxy-5methylisoxazole-4-propionic acid (AMPA) receptors are also heteromeric receptors composed of multiple subunits that are located on postsynaptic membranes. When the GluR2 subunit is present in the receptor, AMPA receptors gate primarily sodium. However, when the expression of this subunit is low, as it is during early development, AMPA receptors can gate both calcium and sodium. Kainate receptors are located presynaptically and postsynaptically on both neurons and glia cells. They are multimeric receptors that gate sodium almost exclusively. Finally, metabotropic glutamate receptors are G-proteincoupled receptors, consisting of eight different subtypes grouped into three functional groups, which are ubiquitously located on presynaptic and postsynaptic terminals (37,38) (see Fig. 4). During development, there are both quantitative and qualitative changes that tip the balance between excitation and inhibition to favor excitatory systems during the early postnatal period. Fig. 5 summarizes the work of a number of laboratories on the ontogeny of receptor deEpilepsia, Vol. 46, Suppl. 7, 2005
velopment in rat brain from birth to adulthood (39–44). One can see that there is rapid growth of both NMDA and AMPA receptor function during the first two postnatal weeks and that peak functional levels overshoot those seen in adulthood by approximately 50%. This sets the stage for the functional predominance of excitation in the neonatal brain. In addition, there are qualitative changes within individual receptor populations that further enhance excitatory neurotransmission (37,38). During early development: •
•
•
•
Expression of the primary glutamate transporter, GLT1, is very low. Hence the postsynaptic response is potentiated due to slower clearance of glutamate from the synaptic cleft (45) NMDA receptors have unique subunit compositions that differ from those of the adult and confer different functional properties. In general, they are more permeable to calcium and have less magnesium blockade. NMDA receptors also depolarize more easily and the resultant excitatory postsynaptic currents are longer in duration (46–49) AMPA receptors also gate more calcium during early development because expression of the GluR2 subunit is low. AMPA receptors also desensitize more slowly and stay open longer, thus potentiating postsynaptic transmission (50,51) Metabotropic receptors have increased turnover of inositol triphosphate (IP3P), which enhances their signaling (52)
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FIG. 4. Excitatory neurotransmission. The schematic depicts the presynaptic and postsynaptic location of the glutamate receptor subtypes and their various subunits. NMDA receptors gate both Ca2+ and Na+ , have a voltage-dependent Mg+ block, and require glycine as a coagonist. AMPA receptors gate mostly Na+ and Kainate receptors gate Na+ exclusively. GluR, glutamate receptors; cAMP, cyclic adenosine monophosphate; PI, phosphoinositide; mGluR, metabotropic glutamate receptors; EAA, excitatory amino acid; KA, kainite receptor; GLY, glycine; PIP2 , phosphatidylinositol 4, 5 biphosphate; PLC, phospholipase C; NMDA, N-methyl-D-aspartate receptor; AMPA, β-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor [for review see Johnson 1996, Raol et al., 2001 (37,38)].
•
Taken together, the results from multiple studies support the hypothesis that the increased seizure susceptibility of the immature brain results, at least in part, from the slow maturation of GABAergic inhibition relative to glutaminergic excitation.
DO EARLY-LIFE SEIZURES ALTER NORMAL PATTERNS OF NEUROTRANSMITTER RECEPTOR DEVELOPMENT AND, IN SO DOING, INCREASE SEIZURE SUSCEPTIBILITY LATER IN LIFE? Young children who experience prolonged or repetitive seizures have an increased risk of developing epilepsy. Emerging evidence suggests that early-life status epilepticus can alter the function of both excitatory and in-
FIG. 5. Maturation of NMDA, AMPA, and KA receptors in rat brain from birth to adulthood [adapted from Sanchez et al., 2001 (59); see also Tremblay et al., 1988 (39); Insel et al., 1990 (40); Miller and Ferrendelli, 1990 (41); Sans et al., 2000 (43)].
hibitory neurotransmitter systems in the brain, resulting in increased hippocampal excitability and lower seizure thresholds. The lithium-pilocarpine (Li-Pilo) model of status epilepticus has been used to study the effects of earlylife seizures on the physiological and functional development of inhibitory GABAergic receptors. In this model, prolonged seizures are induced by giving rats a single intraperitoneal injection of lithium chloride followed by pilocarpine 24 h later. For comparative purposes, experimental treatments are induced in the early postnatal period (postnatal day 10 [P10]) and in adulthood. Study results have demonstrated that early-life status epilepticus produces profound long-term changes in GABAA receptor expression and function in hippocampal dentate granule cells that are opposite to those seen in the adult (53–56). LiPilo seizures induced on P10 produced an overall two-fold increase in GABAA receptor expression and a selective increase in the α1 subunit when the animals reached adulthood (see Fig. 6 and 8). These alterations correlated with functional changes in the expected direction, exemplified by an enhancement of zolpidem-induced augmentation of GABAergic activity. Although these animals were more susceptible to kainic acid-induced seizures in adulthood, they did not show any evidence of spontaneous seizures. In contrast, α1 subunit expression decreased in adult rats following pilocarpine-induced seizures and was associated with a concomitant reduction in the sensitivity to zolpidem augmentation and increased sensitivity to zinc inhibition (56) (see Fig. 7). Furthermore, 100% of these adult rats developed severe epilepsy within a mean period of 4 days after status epilepticus induction (53,56). These findings Epilepsia, Vol. 46, Suppl. 7, 2005
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FIG. 6. Neonatal rats exposed to Li-Piloinduced status epilepticus on P10 (P10SE) show increased total GABAA receptor (Total GABAR) expression in adulthood (A) with a selective increase in the α1 subunit (B) and an enhancement of zolpidem-induced augmentation at concentrations of 100 nM and 300 nM (C). Asterisks denote ∗ p < 0.05, ∗∗ p < 0.01 compared to na¨ıve; #p < 0.05 ##p < 0.01 ###p < 0.001 compared to lithium controls [from Zhang et al., 2004 (53); Hsu et al., 2003 (55)].
suggest that the level of expression of the GABAA receptor α1 subunit may be predictive of the propensity to develop seizures in later life (see Fig. 8). Specifically, high expression of the α1 subunit in hippocampal neurons of the dentate gyrus may be associated with decreased hippocampal excitability and long-term resistance to epilepsy. If future studies confirm this hypothesis, regulation of α1 subunit
levels could represent a new therapeutic target for the prevention or treatment of epilepsy. Early-life seizures also appear to affect the development of excitatory neurotransmitter systems in the brain. Of particular relevance is the nearly two-fold reduction in GluR2 subunit expression that has been observed in the dentate gyrus of adult rats that were subjected to Li-Pilo
FIG. 7. GABAA receptor changes observed in hippocampal dentate gyrus after status epilepticus induced by pilocarpine injection in adulthood. Left: Relative expression of the GABAergic subunits found in single dentate granule cells (DGCs) from control and epileptic rats. [from Brooks-Kayal, et al., 1998 (56); ∗ p < 0.05 ∗∗ p < 0.01. Reprinted from Nature Medicine Copyright 1998, with permission from Elsevier. Right: GABA-evoked responses observed in DGCs were considerably higher in epileptic rats at each of the concentrations tested. In epileptic rats, the mean percent augmentation of the 10 µM GABA response by 100 nM zolpidem was decreased to 18% of controls and the mean percent blockade by 100 µM zinc was increased to 171% of controls. Asterisks (∗∗ ) denote significant difference between groups [from Brooks-Kayal et al., 1998 (56)] ∗ p < 0.05, ∗∗ p < 0.01.
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FIG. 8. Status epilepticus produces agedependent changes in GABAA receptor subunit expression and seizure susceptibility in rats [from Zhang et al., 2004 (53) and Brooks-Kayal, et al., 1998 (56)].
seizures on P10 (54). The presence of high levels of GluR2 subunit expression in normal adult rodents makes AMPA receptor channels less permeable to calcium, thus decreasing channel conductance and reducing neuronal excitability. Low levels of GluR2 subunit expression, therefore, could cause an increase in calcium permeability, higher channel conductance, increased excitability, and hence greater susceptibility to seizures. Indeed, rats that were subjected to Li-Pilo seizures at P10 had lower levels of GluR2 subunit expression and lower thresholds to kainicacid-induced seizures. However, they did not display spontaneous seizures in adulthood. Transient global hypoxia induces prolonged seizures in 10-day-old rats and increases the susceptibility to epilepsy in adulthood (57,58). Therefore, this model provides a good simulation of neonatal hypoxic encephalopathy in man, an early-life insult that is known to be associated with an increased risk of epilepsy in later life. Using this model, Jensen et al. and Sanchez et al. have shown
that GluR2 mRNA levels are significantly reduced in the CA1 region of the hippocampus 48 h after hypoxia (see Fig. 9)(59). After 96 h, there is a concomitant reduction in GluR2 protein expression and an increase in AMPA-mediated calcium permeability, indicating that there is an increase in the number of calcium-permeable AMPA receptors in pyramidal neurons from immature rats exposed to hypoxia-induced seizures compared with age-matched controls. Additional studies have demonstrated that the hypoxia-induced seizures and the subsequent development of epilepsy can be blocked by topiramate, an anticonvulsant drug that works by attenuating AMPA receptor activity (60,61). This suggests that AMPA receptor blockade may be a unique target in the immature brain that can be used both to treat neonatal seizures as well as to protect against their long-term consequences. In addition to their effects on neurotransmitter receptor systems, early-life seizures affect a number of other
FIG. 9. Hypoxia-induced seizures at P10 are associated with decreased GluR2 mRNA in CA1 48 h later (top); by 96 h after hypoxia, there is decreased GluR2 protein expression and increased AMPAinduced divalent cation (Co++) permeability (bottom) [From Sanchez et al., 2001 (59)]. Copyright 2001 by the Society of Neuroscience.
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FIG. 10. Prolonged experimental febrile seizures (FS) at P12 cause persistent hippocampal hyperexcitability (64–67), changes in HCN expression (62,63), and 35% develop spontaneous seizures (66). Top: mRNA levels for HCN1 (left) and HCN2 (right) in CA1 region from control and seizure exposed rats. HT sz = hyperthermic seizures; KA sz, kainate seizures; HT Ctl, hyperthermic controls (rat pups exposed to hyperthermia but seizures were blocked by pentobarbital). Bottom: Western blots demonstrating decreased HCN1 and increased HCN2 protein in hippocampus from rat pups exposed to febrile seizures (FS) compared to controls [from Dube et al., 2000 (64); Chen et al., 2001(71); Brewster et al., 2002 (62)].
critical cell signaling molecules that may ultimately influence the susceptibility of the immature CNS to seizures. For example, recent studies have demonstrated that prolonged febrile seizures on P12 produce a profound, longlasting decrease in hyperpolarization-activated, cyclic nucleotide-gated channel-1 (HCN1) mRNA and simultaneous enhancement of HCN2 mRNA expression in hippocampal CA1 neurons of the rat (see Fig. 10) (62,63). These changes were associated with persistent limbic hyperexcitability and a 35% incidence of spontaneous seizures in adulthood (64–67). Again, this suggests that a variety of receptors and important cell signaling proteins may be permanently altered following early-life seizures. CONCLUSIONS Cumulative evidence suggests that the developmental imbalance between excitation and inhibition may contribute to the increased susceptibility of the immature brain to seizures. Similarly, early-life seizures can disrupt normal activity-dependent patterns of receptor development in both excitatory and inhibitory neurotransmitter systems in the brain. Although some of these changes may be protective, others may enhance limbic excitability and increase the probability that epilepsy will develop at a later time. It is hoped that identification of these molecular changes will provide novel therapeutic targets for the treatment and/or prevention of epilepsy due to early-life brain insults. Epilepsia, Vol. 46, Suppl. 7, 2005
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