The neurobiology of epilepsy is an active field of investigation. Numerous mechanisms of inhibitory and excitatory deregulation have been suggested, and the interactions of these pathways are observed almost ubiquitously. The compromised inhibitory compensation of Gamma-Aminobutyric Acid (GABA) during seizures has been hypothesized to be a direct result of aberrant glutamate metabolism. Recent research has also implicated the role of endocannabinoids in modulating epileptic activity in the hippocampus. A stimulating area of epilepsy research is the examination of different models of circuitry participating in seizure activity. Other physiological changes that occur in the epileptic brain include glial composition changes and synaptic modification. Together, these findings provide a better understanding of the neurobiology of epilepsy, and could lead to improved management and cures.
Introduction
Epilepsy is a neurological disorder characterized by unprovoked, spontaneous seizure activity due to abnormal, excessive, or synchronous neuronal activity. It can be caused by specific cortical deformities such as lesions, tumors, developmental malformations, or other neurological complications such as Alzheimer’s or cerebral palsy, or be of idiopathic origin. Epilepsy and seizures affects more than three million people in the United States, with approximately 200,000 new cases being diagnosed each year (1). It is one of the most frequent disorders among children, and causes impairments in psycho-motor and cognitive development ranging from mild to severe (2). Patients with epilepsy suffer from debilitating social problems and stigmas, as well as increased risk for co-morbid psychiatric disorders such as depression, anxiety, and suicidality (3).
The majority of seizure disorders are treated with administration of anticonvulsive or antiepileptic drugs (AEDs), while a smaller percentage of patients receive neurosurgery or surgical implants to control their seizures. The treatment of epilepsy is varied and tailored to each patient’s situation. Understanding the underlying neural networks of epilepsy is crucial in helping physicians assess the appropriate treatment for a better outcome. Seizure disorders affect a variety of brain pathways and mechanisms that interact in different ways. A wide range of receptors and neurotransmitters are modulated in epileptic patients, including GABA, glutamate, and endocannabinoids. Investigating the precise functions of these neural components in seizure activity will help formulate more specific and efficient AED treatments so that side effects can be reduced and seizures better managed. Here, I will review the recent findings of the role of different mechanisms and receptors in seizure activity, followed by network and structural changes that accompany the epileptic brain.
Mechanisms of Epilepsy
Different inhibitory and excitatory pathways undergo modification and contribute to epileptogenic activity. Deregulation can occur at the synaptic level, and may result in a change in receptor expression, neurotransmitter reuptake, or binding affinity.
The Glutamate-Glutamine Cycle Modulates Neuroinhibition
GABA and glutamate imbalance is a key cause of the hyper-excitation and hyper-synchronization of neurons, which lead to synaptic changes. An experiment examined the changes in GABA and glutamate neurotransmission in rats with generalized absence seizures, which are seizures that spread to the entire cortex and occur frequently in human populations. In vivo uptake of glutamate was found to be compromised in the cortex of epileptic rats compared to controls, while extracellular levels of GABA and glutamate were unchanged in the cortex and thalamus (4). This finding suggests a dysfunction in glutamate transportation, which may cause spike wave activity indicative of brain dysfunction in the cortex of rats with absence epilepsy.
The regulation of extracellular glutamate levels is important because sustained activation of glutamate receptors results in excitotoxicity and leads to neuron death. Glutamate transporters EAAT-1 and EAAT-2 are found in astrocytes and take up extracellular glutamate against the concentration gradient to prevent CNS injury (6). Near glutamatergic synapses, astrocytes express an enzyme called glutamine synthetase (GS) which converts glutamate to its inactive form, glutamine. Glutamine is then shuttled into the neuron through a system A transport system, protecting the cell from toxicity (7). These two mechanisms serve to regulate deleterious glutamate activation. Inside the neuron, glutamine can be converted back to glutamate by mitochondrial glutaminase and stored in vesicular glutamate transporters (VGLUT) until release (6). The glutamate-glutamine cycle therefore functions as a crucial neuroprotective mechanism to prevent glutamate-related excitotoxicity and oxidative stress.
The excess of excitatory activity is mainly a glutamatergic event, and consequently extracellular glutamate is elevated in hippocampi of patients with intractable mesial temporal lobe epilepsy (MTLE). Extracellular glutamate levels also increase during a seizure and the glutamate is cleared more slowly than usual afterwards. In a recent study by Eid et al., the accumulation of extracellular glutamate in MTLE hippocampi is proposed to be due to a deficiency in astrocytic GS enzyme levels.
The researchers performed enzyme activity assays and found that the enzyme catalyzing glutamate synthesis, phosphate activated glutaminase (PAG), was increased in the epileptic hippocampus (9). This finding suggests that there is increased potential for glutamate production resulting in increased excitation in the MTLE hippocampus. What needs to be further investigated is what controls the changes in GS and PAG. Speculations can be made about the upstream involvement of transcription and translation, and studies examining the expression patterns of mRNA for these two enzymes need to be performed to confirm this.
Glutamate has an additional role in inhibitory transmitter regulation. Glutamate and GABA inter-terminal levels exist in equilibrium, and the glutamate-glutamine cycle also functions as a regulator of synaptic GABA release. In neurons, glutamate can be decarboxylated by glutamic acid decarboxylase (GAD) to generate GABA, which is packaged into vesicles. Studies have shown that disturbing the glutamate-glutamine cycle results in decreased hippocampal GABA reserves, and that GS levels are significantly decreased in epileptic human hippocampi (8). These data indicate that glutamate and GABA participate in the convergent regulation of excitatory and inhibitory networks.
In a recent experiment by Liang and Coulter, the deficiency of the glutamate-glutamine cycle in rats with temporal lobe epilepsy (TLE) was hypothesized to compromise GABAergic inhibitory synaptic efficacy. They performed whole-cell patch recordings from the pyramidal neurons of CA1, a region of the hippocampus containing several output pathways. They found that inhibitory post-synaptic currents (IPSCs) were much slower and smaller in epileptic rats, which was consistent with a decrease in synaptic GABA concentration as well as vesicular GABA content (5). The decrease in vesicular GABA release was thought to be due to the compromised function of the glutamate-glutamine cycle. To test this speculation, they examined changes in IPSCs induced by extracellular stimulation using a patch pipette and measuring synaptical GABA release. In the presence of a glutamine uptake inhibitor, a reduction in synaptic strength of IPSCs was observed in control CA1, but not in neurons from rats with TLE. They concluded that extracellular glutamine was not used by the cell to make GABA in inhibitory synapses of epileptic rats. Interestingly, the administration of exogenous glutamine restored inhibition in CA1 of epileptic animals, but had no effect on controls (5). Combined, these results show that lowered vesicular GABA synthesis and release are caused by a deficiency in the glutamate-glutamine cycle. Therefore, glutamate affects the efficacy of the GABA inhibitory response during seizures, and this compromised GABA release fails to reduce neuronal activity in a timely fashion.
Endocannabinoid Hypotheses
The endocannabinoid system is an important neuromodulatory system, and is involved in the regulation of excessive excitatory neuronal activity. It consists of cannabinoid receptors, which bind endogenous lipid ligands. The type 1 receptors (CB1) are G-protein coupled seven-transmembrane domain proteins mainly expressed in the forebrain, and their activation by endocannabinoids activates a cascade of functions including inhibition of neurotransmitter release, long-term plasticity, and control of excitatory activity (10). In the hippocampus, amygdala, and neocortex, CBI receptors are colocalized in the terminals with GABA receptors, but in the cerebellum and striatum, are found in glutamatergic neurons (11). Therefore, endocannabinoids can either have either have inhibitory or excitatory effects depending on the receptor which they activate and the brain region on which they bind.
In a current study by Bhaskaran and Smith, changes in the synaptic composition of cannabinoid receptors in the dentate gyrus of a pilocarpine-injected mouse model were examined. After inducing status epilepticus in mice, mossy fiber sprouting and spontaneous firing of neurons were found in the hippocampus, and especially in the dentate gyrus. These fibers are bundles of axons projecting from granule cells through the hilus, and connecting to the pyramidal cells in CA3 (18). The reorganized tissue contained CB1 receptors as well as a class of capsaicin-binding villanoid receptors (TRPV1), which integrate inflammatory stimuli and play an important role in the pathophysiology of sensitization (11). Anandamide is an endocannabinoid which can activate both CB1 and TRPV1 receptors and was used to explore the potential therapeutic advantages of endocannabinoids in the treatment of TLE (12). Anandamide was found to diminish excitatory post-synaptic currents (EPSCs) in the altered dentate gyrus of seizure-induced mice, but sometimes it enhanced excitatory responses. When CB1 receptors were blocked with AM251 antagonist, administering anandamide caused an increase in EPSCs. However, when TRPV1 receptors were blocked with capsazepine, anandamide had the opposite effect, and suppressed neuronal activity. When CB1 was activated by a receptor-specific agonist, EPSCs decreased in duration and strength, and when TRPV1 receptors were activated by capsaicin, EPSCs were enhanced (12). These results indicate that anandamide can have either an inhibitory or excitatory effect depending on the receptor it stimulates.
Another recent experiment by Ludanyi et al. found that epileptic hippocampi exhibited a down-regulation of the neuroprotective CB1 receptor. Quantitative PCR measurements showed that mRNA levels of the CB1 receptor in epileptic hippocampi were one third of the value in controls. Similarly, the cell surface expression of cannabinoid receptor-interacting protein was decreased. Finally, the researchers performed immunoblotting studies and showed that the density of CB1 was decreased in the hippocampi of epileptic patients, most robustly in the dentate gyrus (13). That this experiment was performed in human samples should be of note. Control hippocampal tissue was obtained from post-mortem samples from subjects with no known neurological disorders, and epileptic tissue was obtained from surgically resected patients with intractable TLE. Although more human studies are needed, results from post-mortem samples may not have significant in vivo implications. The composition of tissue changes slightly after death, and previous studies even found a small decrease in the post-mortem localization of the astroglial transporter protein in TLE (14). Therefore, more experiments exploring endocannabinoid pathways need to be made in both mice models and humans in vivo to provide more convincing support.
Endocannabinoids have been associated in the regulation and modulation of various physiological behaviors, including eating, anxiety, pain, and aversive memory extinction (11). Their potential therapeutic use in neuroprotection, specifically down-regulation of excitatory brain activity in epilepsy deserves further investigation. Future studies should examine the role of plasticity in the endocannabinoid system and determine if any of the harmful synaptic reorganization and neuronal damage due to aberrant excitatory activity can be reversed.
Network and Structural Changes
Seizures are the outward expression of abnormal brain activity, which can be seen in irregular EEG patterns. Several physiological changes can be observed in the hippocampus of epileptic patients, including neuronal degeneration, mossy fiber sprouting, basal dendrite formation on granule cells, aberrant synaptogenesis, astriogliosis, and microglial activation (17). Here I will examine the findings on connectivity changes caused by seizures, and observe how the brain as a whole behaves in epilepsy.
Network Topology on Seizure Spread
The complex neural networks involved in epileptic activity are not well understood. Standard explanations for balanced neural activity rely on groups of inhibitory neurons that suppress excitatory neuron firing. Kaiser and Hilgetag investigated the effect of network topology on preventing large-scale seizure spread, specifically the propagation of signal through hierarchically clustered neurons. They created a model where neurons were represented as binary nodes which were activated by a certain number of surrounding active neurons, and were deactivated over time. This basic model imitates the cerebral cortex network which spans several levels of organization, and was compared to isolated and random circuits of the same size. They found that higher degrees of cluster connectivity resulted in more frequent and complete spreading activity, which was not true for isolated and random models. In contrast, hierarchical clustering prevents large-scale activation, and provides stable neural activity (15). Inhibition at the hierarchical level would therefore provide efficient regulation of neural activity. These computational models can have significant applicational value. Additional research is needed to determine if epileptic patients do in fact have higher inter-cluster connections, and if this is the cause of widespread and sustained electrical activity during a seizure. In addition, the role of inhibitory neurons needs to be factored into the model to provide a more complete and accurate understanding of the neural circuits that underlie epilepsy.
Another recent study by Schiller and Cymerblit examined the network dynamics leading up to the development of seizures, and possible ways to predict them. They conducted multi-electrode single unit recordings for hippocampi of rats with induced pilocarpine and picrotoxin epilepsy (16). They recorded action potentials of individual neurons and assessed the synchrony of the network and firing patterns during seizures. Changes in EEG patterns were observed 15 minutes before seizure onset, which can be separated into two phases. In the early phase, there is a slowing of activity, and fewer neurons fire together and at the same time. In the later phase, neurons tend to fire in synchronous bursts, causing sharp waves in the EEG recordings, suggesting possible activations of positive feedback loops (16). These findings suggest that in pharmacologically-induced seizures, specific and predictable changes in firing patterns of hippocampal neuron populations occur during the pre-ictal state. Understanding the network dynamics responsible for seizure initiation helps detect seizures before they start, so that timely treatment can be given.
Astrocytic and Microglia Changes
Glial activation is known to occur after brain injury, trauma, or seizures, and play a critical role in neuronal degeneration and survival. Significant changes in glial composition take place in the epileptic brain of pilocarpine-injected rats, which closely mimics the human condition (16). Shapiro et al. conducted a study examining the precise temporal changes in epileptic hippocampi glial composition of rats over a period of eight days. They labeled astrocytes with GFAP and labeled microglial cells with IBA-1. One day after seizures, an increase in labeled astrocytes was evident in specifically the hilus of the hippocampus, followed by a transient decrease over two to five days. Amounts of labeled microglia increased initially in the hilus, CA1, and CA3 regions, which progressively decreased over a period of three days, then increased again at day eight (18). These results indicate that astrocytes and microglia exhibit specific temporal and spatial changes after pilocarpine-induced seizures.
More precise descriptions of these changes, such as specific cell counts, are needed for a better understanding of glial mechanisms involved in epilepsy. The release of cytokines activates gliosis during neuronal damage, and recruits astrocytes and microglia to the site of injury to induce apoptosis and neuronal repair (17). The signals for glial activation need to be elucidated so that better pharmacological management can be used to exploit their many reparative roles.
Conclusion
Combined, the recent findings help us formulate a better understanding of the causes and effects of epilepsy. We are still at the early stages of investigation, and recent technology such as fMRI and other imaging techniques has helped advance our knowledge of the disorder in recent years. However, for future research, more standards need to be established so that results can be compared across studies. For example, results could be less variable across experiments if a consistent protocol were developed for the induction of pilocarpine seizures in rat or mouse models. The general procedure for making rat or mouse models of epilepsy is to inject pilocarpine locally in the rat brain, causing status epilepticus, and then stopping SE by administration of an anticonvulsant. This causes the animal to develop epilepsy, which is characterized by the occurrence of spontaneous and unprovoked seizures (19). Too many variables in procedure exist presently, such as length of drug administration, amount in each injection, length of time between injections, and length of time to wait before stopping SE. Data could be compared across experiments with more confidence and results could be replicated more easily if most of these variables were eliminated.
In addition to aiming for less variable data acquisition, more in vivo studies should be conducted. While insightful knowledge can be gained from examination of tissue and single-cell recordings, what occurs in a live model can differ significantly from the in vitro study. Most studies focus on the hippocampus as a key structure involved in epileptic activity and possibly epileptogenesis. However, additional experiments that investigate other afferent and efferent circuitries and their role in propagation of seizures are needed. Research on neighboring brain structures involving the limbic and thalamic systems can clarify the methods by which a seizure can spread to the whole brain and compromise consciousness. Genetic studies are worthy of conducting, and may provide invaluable information about the predisposition and prevalence of epilepsy among human populations. The influence of environmental factors on the neurobiology of epilepsy is also an area of potential investigation. The future of patients with seizure disorders depends greatly on further research leading to improved clinical management.
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