Wednesday, August 4, 2010

Dealing with Epilepsy – Establishing Better Treatment Through Co-Therapies

Treatment of epilepsy is interesting because epilepsy affects up to 1% of the world population1,2 yet there seems to be very little publicity surrounding the disease versus other neurological conditions like Alzheimer’s Disease and Parkinson’s Disease. In fact the only time epilepsy is discussed in a public forum is when some celebrity is afflicted. Interestingly still is that based on epidemiological studies from the U.S. about 3% of all people living to 80 years old will suffer from some form of epilepsy.3 The reason for this lack of attention may be that although epilepsy does negatively influence quality of life for those afflicted with it, it has a low associated morality rate. Also unless an individual is currently experiencing a seizure it is difficult to identify individuals that suffer from epilepsy, thus there are less visual cues and experiences to spur concern. Although anti-epileptic medications (AEMs) have proven useful at managing seizures in most patients, most of them induce a wide variety of negative side effects making their administration less desirable. Surgical options to relieve seizure activity are a mixed bag where sometimes surgery is successful, sometimes it is not successful and sometimes the results are unclear due to changes in seizure frequency and intensity. Thus it would be useful to review the general information regarding epilepsy in order to identify a more definitive pathology generating a better understanding of epilepsy and the ability to devise more sustainable treatments.

Although approximately 40 different types of epilepsy have been identified, this post will focus on temporal lobe epilepsy (TLE) seeing that it is the most common form of epilepsy.3 TLE is a type of focal epilepsy (that which creates focal seizures). Focal seizures are also referred to as partial seizure and describe the origins of a seizure in a small group of neurons in a specific location in the brain.3 For TLE seizure onset is thought to originate in one or both temporal lobe(s). There are two main types of TLE, medial TLE where seizure origin is in the hippocampus, parahippocampal gyrus and amygdala and lateral TLE where seizure origin is in the neocortex.3 With regards to seizure activity, those suffering from TLE experience simple partial seizures (SPS) and complex partial seizures (CPS) and secondarily generalized tonic-clonic seizures (SGTCS).

Of the three major seizure types experienced by those suffering from TLE, SPS are the least severe. SPS commonly originate in the hippocampus or amygdala. Incidentally the hippocampus is the location of the brain that receives the most research focus with respects to TLE. In SPS consciousness is not lost, instead the patient may suffer from various sensations like déjà vu or amnesia during the time of the seizure.3 SPS are not the stereotypic seizures where the sufferer writhes on the floor as his/her body shakes uncontrollably, but instead sensations are felt which do not significantly influence the ability of the sufferer to interact with his/her environment. For example a partial seizure may begin with simple jerking of a hand that later evolves into jerking of the entire arm for a very brief moment.

CPS are the second stage of seizures, worse than SPS but are still not emblematic of the stereotypical seizure even though they can impair consciousness at some level.3 CPS originate as SPS, but in some cases of epilepsy due to the progression of neural damage the seizure spreads impacting a greater area of the temporal lobe. This influence typically results in changes in speech tone and/or rate, automatic perceived unconscious movement and motionless staring among other things.3

Although disruptive the real concern with CPS is their evolution into SGTCS. SGTCS are the generic stereotypical seizures that most people typically recognize as the calling card of epilepsy where an individual commonly falls to the ground due to stiffening of the arms, legs and mid-section later resulting in wild jerking of the appendages. Both CPS and SGTCS cause amnesia for the duration of seizure, similar to SPS, but the amnesia is longer lasting and more substantial. Symptom progression in a SGTCS begins with expansion of the seizure to where it influences both brain hemispheres, so the results of the neural activity influences all motor reflexes. Then the seizure enters the tonic phase where all of the patient’s limbs become rigid leading to loss of balance and the inability to remain standing.3 Finally the seizure progresses into clonic phase where all major extremities begin seizing.3 SGTCS are also sometimes referred to as grand mal seizures. Note that SGTCS can occur outside the evolution of a partial seizure, but because TLE based seizure activity involves partial seizures developing from a seizure focus other means to facilitate SGTCS will not be discussed further. Overall the phases in the development of a partial seizure can be generally divided into the interictal period to neuronal synchronization to seizure spread to secondary generalization.3

Clearly there is a neurological deficiency which leads to the development of epilepsy and its associated seizures. The best way to try to understand the origins and prognosis of this deficiency is to review how the normal function of the brain and where the breakdown occurs in TLE. Note that epilepsies are defined in a minimum context by the reoccurance of seizure activity without a single sensory cause (i.e. looking at a flashing light).

In humans, the hippocampus is found within the medial temporal lobe.3 The primary role of the hippocampus is to govern short-term memory storage and aid spatial navigation. One of the immediate problems is that although there is general scientific consensous regarding that primary role of the hippocampus, the methodology behind how it accomplishes that role is still debated. Fortunately the cause of TLE is not predicated by short-term memory loss or loss of spatial navigation outside the occurrence of a seizure, lessening the need to navigate that debate.

To understand the occurrence and progression of TLE it is important to review the order and operation of the neuronal connections. The first important region is the dentate gyrus, which is technically not part of the hippocampus, but does contain granule and mossy fiber cells that provide input to the Cornu Ammonis (CA) regions, most specifically CA3.3 The entire CA region is divided between four specific sections aptly named 1, 2, 3 and 4 traveling in descending order in distance from the dentate gyrus. The CA region as a whole is commonly referred to as the ‘hippocampus proper’ where the CA1 and CA3 regions are considerably larger than the CA2 or CA4 regions. Each CA section is filled with densely packed pyramidal cells similar to those found in the neocortex.3 The CA1 region feeds into the subiculum which leads to the presubiculum and parasubiculum. Finally these regions feed into the entorhinal region, which feed back into the dentate gyrus forming a form of feedback loop.3

As mentioned a large amount of evidence suggests that TLE seizures are thought to originate in the mesial temporal lobe, which correlates well with the atrophy and cell loss that are seen in the mesial temporal lobe in epilepsy patents.2,3 The most significant damage in TLE seems to occur in the CA1 region with associated damage in the CA3 region and the hilus of the dentate gyrus.3 Popular opinion suggests that a hyperexcitable dentate gyrus is the principle cause of TLE. There is little damage in the subiculum, but the subiculum may be the most important region with respects to developing epileptic capacity. The generic strong neuronal cue before seizure onset in the seizure focus is the development of a synchronized electrical response called the paroxysmal depolarizing shift (PDS). The PDS is a 20-40 mV depolarization event lasting (50-200 ms) involving multiple action potentials.3

Not surprisingly the most pressing issue surrounding epilepsy is the role of γ-amino butyric acid (GABA), which is the chief inhibitory neurotransmitter in the brain and is a major component responsible for determining the frequency and strength of neuron firing. GABA operates as an inhibitory neurotransmitter by opening chloride permeable ion channels after binding to GABA receptors (normally GABAA) on post-synaptic neurons. When these channels open there is typically an influx of chloride ions into a neuron based on the existing electrochemical gradient, which reduces the membrane potential of the neuron reducing the probability that the cell depolarizes and fires an action potential. The inhibitory action of GABA is imperative to ensure a steady and structured rate of neuronal firing.

Based on its inhibitory role GABA plays an important part in the question of epilepsy pathology and occurrence, but a wide variety of confusing empirical information cloud the specifics of that role. Regardless of this conflicting evidence one of the major theories that has been proposed to explain the progression of epilepsy is the Dormant Basket Cell Hypothesis (DBCH). DBCH is one of the most popular and principal theories trying to explain the pathology behind epilepsy. The idea behind DBCH was proposed by Robert Sloviter in an effort to explain the occurrence of reduced inhibitory inputs when most of the GABAergic basket interneurons appear morphologically intact (the neurons do not appear dead due to structurally sound synapses).4,5,6 DBCH presumes that the loss of excitatory inputs for GABAergic basket interneurons, largely due to the death of hilar mossy fiber cells in the dentate gyrus, reduces the firing probability of these GABAergic neurons reducing the total concentration of GABA released into the synapse.4,6 This reduction in GABA concentration reduces the probability of inhibition of granule cells in the dentate gyrus, which continue to fire action potentials leading to seizure activity due to the excess excitation.

Unfortunately like with almost all theories the DBCH has its detractors, which question whether it is just a theory that was created to match some empirical data. One of the principle arguments of the detractors is that DBCH associates the loss of mossy fibers as one of the chief culprits behind excitatory input loss and similar to other information in epilepsy there appears to be contradiction in mossy fiber survival. Some argue that mossy cells do not die at levels required to facilitate dormancy7 where others argue mossy fiber death at much higher levels, more than enough to silence enough GABAergic neurons.8,9,10 Realistically it is likely that a significant number of mossy fiber cells do die, but whether or not their deaths are enough to matter is another issue altogether.

For instance there is also a second issue with mossy fibers in that some believe that they are not primarily responsible for providing the majority of the excitation input to interneurons in the dentate gyrus. There are reports that mossy fibers only provide an insufficient level of excitatory input to begin with and their loss is immaterial to the onset of epilepsy. These opponents of DBCH believe that most of the glutamatergic input to basket cells arrive from the entorhinal cortex (EC), from mossy fiber collaterals, granule cells in the fascia dentata and through feedback mechanisms from CA3 pyramidal cells.11,12,13,14,15,16 If mossy fibers do not provide sufficient input to the dentate gyrus then their deaths would be of little consequence to the action of basket cells in the dentate gyrus.

For the moment assume that enough mossy fiber cells remain intact or they are not the principle excitatory input provider to GABAergic interneurons. The acceptance of either one of these two suggestions force a re-examination of the lost GABAergic interneurons in the dentate gyrus because if mossy fiber death is not sufficient or relevant enough to ‘mute’ enough basket cells then GABAergic interneuron death may play a larger role in epilepsy than the DBCH credits.

Various evidence has identified a number of glutamatergic neuron types that are killed in and after status epilepticus (SE) which project into the dentate gyrus including granule cells,17 layer II EC neurons,18 GluR2-positive hilar neurons9 (a receptor commonly expressed by mossy fibers) and CA3 pyramidal cells19 which all have synapse connections with basket cells.15,16,20 Incidentally the death of the CA3 pyramidal cells may be the most interesting of the three with regards to the total excitatory input of the basket cells because their input typically operates in a feedback nature, which would briefly increase due to hyperexcitability (assuming that excitotoxicity is responsible for CA3 pyramidal cell death) then drop-off as the CA3 cells die perhaps limiting their overall influence in the total progression of epilepsy.

With the level of confirmed glutamatergic neuronal death not just isolated to the mossy fibers, it would make sense to expect a reduction in excitatory input frequency in the dentate gyrus, a result that has been demonstrated empirically.21 Although there is little objection to the notion that excitatory input frequency drops over in the short-term, there is question to whether or not excitatory input frequency recovers over time. Some argue that resultant mossy fiber sprouting can restore excitatory input frequency8 whereas others do not see any significant recovery in their results.21,22

Regardless of the excitatory inputs lost over the progression of epilepsy, it is also important to investigate the extent of GABAergic interneruon loss. Two of the principle proteins expressed by GABAergic interneurons that are affected by the progression to epilepsy are parvalbumin (PV) and calretinin (CR). Sometimes these elements are referenced as parvalbumin-immunoreactive (PV-IR) and calretinin-immunoreactive (CR-IR).2 Parvalbumin is a calcium binding protein, which is thought to play a role in calcium sequestration and sensitivity.3 Calretinin is also a calcium-modulating protein that influences calcium dependent neural processes.3 PV is expressed in both basket and chandelier interneurons.2 PV is also expressed by bistratified and oriens-lacunosum moleculare (O-LM) cells, but those cells do not appear to be overly relevant to epilepsy. Note that basket interneurons typically have an axon that innervates the soma of the input cell and chandelier interneurons typically have an axon that innervates the axon initial segment (AIS) of the input cell.23 Also PV-IR interneurons also innervate the perisoamtic region of their respective input cells as well as the dendritic region.24,25,26

Axons that innervate to the periosomatic region not surprisingly drive perisomatic inhibition which limits action potential firing where axons that innervate to the dendritic region limit synaptic integration, which if done in a large enough quantity can limit action potentials.2 Total dendritic length and diameter is largest for PV-IR where CR-IR are smallest among the four principle expression elements in epilepsy (the other two will be introduced shortly).27 Due to their increased dendritic length, the density of inputs and total number of afferent synapses are several times larger (4-8 times) for PV-IR any other interneurons, but due to these connections PV-IR receives the highest ratio of excitatory-inhibitory inputs (93.6%-6.4%).27 Pyramidal cells tend to form single synaptic contacts with their target elements,28,29 so excitatory input from pyramidal cells to a given neuron matches the number of converging pyramidal neurons. Interneurons tend to form multiple contacts with their targets across multiple targets,30,31 which places more importance on each inputting interneuron. Basically if one interneuron and one excitatory that innervate the same neuron die the receiving cell will typically lose more inhibitory potential then excitatory potential even though the same number of input neurons were lost. This structure supports the notion of more active hippocampal basket cells than other interneuron subsets.30,32

Finally PV-IR interneurons seem to play a role in aiding the synchronization of principle neuron populations during network oscillations.30 CR-IR interneurons are responsible for innervating other interneurons, are thought to synchronize their response to eliminate synchronous excitatory firing and appear sensitive to epilepsy in both animals and humans.32,33 Overall dentate basket cells expressing parvalbumin account for approximately 8-9% of all GABAergic interneurons in the dentate gyrus34 and recall that they receive the highest number of synapses among the different interneuron types in the rat hippocampus while receiving the smallest proportion of GABAergic input.27

Another important protein that is commonly expressed in GABAergic neurons lost during the progression of epilepsy is somatostatin. Somatostain serves two major known roles in the body, a polypeptide hormone and a peptide neurotransmitter and is generally responsible for inhibiting growth hormone secretion and other elements in the endocrine system. Somatostain expressing interneurons typically account for approximately 25% of all inhibitory interneurons in the dentate gyrus of rats and their axon collaterals concentrate in the middle and outer molecular layer synapse with dendrites of granule cells.35,36,37,38 Due to their GABAergic nature and spatial positioning somatostain expressing interneurons are effective inhibitors of granule cells at sites of perforant path input, which is the major afferent of the dentate gyrus,39 however, one must remember that somatostain inhibition is typically still weaker than PV-IR neuronal inhibition due to the somatic vs. dendritic mechanism of the inhibition.

Finally what might be the most important, but also most mysterious protein in epilepsy progression is Calbindin D28K (CB). CB-expressing cells have axonal arbors which overlap Schaffer collateral terminals in the dendritic region33 and are thought to have some influence over dendritic calcium spikes in principle neurons.30,33 Also CB-expressing terminals have recently been reported to make symmetrical synapses with the AIS of pyramidal cells in CA1 of the normal human hippocampus implying that some chandler cells could express CB.46 Interestingly there are also questions to how CB expression changes in response to epileptic damage where it decreases in granule cells46,47 and tends to increase in interneurons.48,49,50 This trend in CB expression (increase in interneurons and decrease in excitatory neurons) is not isolated to the dentate gyrus, but seems to extend to CA1 and CA3 regions as well.51 The reason for these changes in the CB expression are currently unknown largely because of the continuing confusion surrounding the role of CB in neurons beyond its role as a calcium buffer and transporter.

Neuropeptide Y (NPY) is another molecule that may play a role in epilepsy development. NPY is a peptide neurotransmitter that plays a role in food consumption and possibly fatty acid storage and is commonly co-expressed with PV or somatostatin. Unfortunately the role of NPY in epilepsy is currently unclear and it may not even play a role at all instead simply being used as a secondary confirmation methodology for the death of somatostatin expressing and PV-IR cells.

During the progression of epilepsy it is believed that PV-IR interneurons are lost in the dentate gyrus,40 the CA1 and CA3 regions,41 most notably in the stratum oriens layer,42 and the subiculum.2 CR expressing interneurons are lost in the subiculum2. Somatostatin expressing interneurons are lost in the dentate gyrus43,44,45 and the CA1 region also in the stratum oriens layer.41 Note neurons in the stratum orines have dendrites that extend into all strata and axons typically projecting into basal dendrites and soma of pyramidal cells within the hippocampus.41

While the deaths of somatostatin and CR expressing neurons are widely accepted in the scientific community the overall vulnerability of PV-IR interneurons is somewhat controversial. Some studies have demonstrated large-scale death of PV-IR interneurons,2,46,52 whereas other studies have demonstrated large-scale survival of PV-IR interneurons,53,54 where another study demonstrated some death and some survival depending on given conditions.54 The three most likely explanations for these contradictory results are: first, an unclear methodology of neuronal death in epilepsy may create a time variance between studies where some studies have not progressed long enough to witness the death of PV-IR interneurons. Second, the type of stimulation applied to facilitate the epileptic condition in either the animal model or the human tissue changes the overall fate of PV-IR interneurons. For example application of the perforant path stimulation (perforant path model for TLE) might not induce significant PV-IR interneuron death,8 but application of the pilocarpine-based stimulation (pilocarpine model for TLE) can induce significant PV-IR interneuron death.44 Third, the difference stems from the ability of an interneuron to express PV under epileptic conditions versus normal conditions. Basically PV expression in a neuron decreases in epileptic conditions, but the neuron itself does not die.

Unfortunately attempts to try to separate PV staining from PV-IR interneuron death have not been without contradiction. One study used Fluoro-Jade B to determine cell death.54 Fluoro-Jade B is a cell fluorochrome that is able to identify degenerating neurons. Although the mechanism is not fully understood, numerous secondary empirical confirmation have classified it as relatively accurate. Fluoro-Jade B labeling identified little PV-IR interneuronal death even after generation of SE.54 However, other studies have focused on how synaptic bouton loss relates to the number of GAD65 expressing perisomatic terminals and that their proportional loss implies significant death of PV-IR interneurons.2,41 Another study suggested, after examining epileptic tissue against control tissue using electron microscopy that at least in the CA1 region basically in non-sclerotic epilepsy there is low PV-IR interneuron loss, but in sclerotic epilepsy there is significant PV-IR interneuron loss.55 To the authors the pattern of death seemed to imply that as long as at least one target remained for a given PV-IR interneurons it was more probable that it would survive than die.55

Some view the question of PV-IR survival as important because if PV-IR interneurons survive in epileptic conditions it is highly probable that they are not functioning at normal levels, so it stands to reason that if excitatory inputs are returned then normal inhibitory operation should commence (one of the base tenets of DBCH). However, others have identified that in addition to the loss of some interneurons there is also some evidence that the surviving PV-IR interneurons develop shrunken somata and shorter dendrites,56 which other types of interneurons fail to develop similar morphological changes.

One of two explanations can be used to explain this retraction behavior; first shrunken somata and dendrites may be interpreted as an intermediate stage before death due to lack of input; second it is a morphological change designed to improve survival in a more excitatory environment; basically a form of protectionism by the brain in an attempt to reduce the probability of excitotoxicity through a reduction of depolarization probability. The correct explanation depends on the total excitatory input received by these basket cells, a reduced amount would favor the first explanation and an increased amount would favor the second explanation. The second option is a possibility because PV-IR neurons can die through excitotoxic mechanisms in the CA1 region.57,58 Either way it is reasonable to assume that these PV-IR neurons with shorter dendrites and shrunken somata will have less opportunity to participate in the inhibitory pathway.

When taken all together it is reasonable to suggest that a significant enough number of PV-IR and somatostatin expressing interneurons are impacted in the dentate gyrus to reduce inhibition to a level that facilitates spontaneous seizure activity. Overall the general debate regarding PV-IR death probably relates back to time, PV-IR interneurons do eventually die just not nearly as quickly as somatostatin expressing interneurons. Regardless the debate between DBCH vs. neuronal death really only matters in the nature of treatment. If neurons die then stem cells would be a more attractive treatment option, but if dormancy were prevalent then implanting some form of electrical pacemaker to stimulate basket cells would prove to be more useful. Note: electrical pacemaker type devices have been attempted at very low experimental populations yielding fair results.

If dormancy is the principal rationality behind the development of epilepsy, why do GABA-enhancement drug therapies work so well? Based on the methodology of DBCH the ability of the GABAergic interneurons to naturally inhibit the granule cells in the dentate gyrus no longer exists because of a lack of excitatory input. However, typical AEM treatments only work to enhance the longevity or effectiveness of naturally synthesized and released GABA. If a neuron is not producing and releasing GABA, due to a lack of excitatory input, the AEM would not be very effective. Therefore, either the dormancy of the GABAergic interneurons must be rather subtle (the loss of the excitatory input is only enough to push the provided inhibitory input just below some required threshold for seizure suppression) or if cells are lost, those cells play a more important role in the maintenance of inhibitory balance than other interneurons, a role that is filled by AEMs.

Clearly the differing results seen from AEMs that focus on augmenting GABA implies other elements that influence the progression of epilepsy. The very fact that GABA drugs work at all support the conclusion that there is a reduced GABA concentration in the synaptic cleft vs. controls. Also there is a lot of evidence to support additional sprouting axons in the collaterals and mossy fibers in both GABAergic interneurons and glutaminergic interneurons. The potential for sprouting makes sense because of brain plasticity, the ability to rebuild lost connections deemed important or at least compensate for them by expanding existing connections to maximize the probability of neurotransmitter binding in an environment with reduced neurotransmitter.

Although the extent of mossy fiber cell death is somewhat debated in epilepsy, mossy fiber sprouting is not. It is almost universally agreed that surviving mossy fiber cells go through a period of dendritic and axonal sprouting in response to the morphological and psychological changes that occur during epilepsy.9,44,59 Other granule cells are also thought to sprout axon collaterals which travel into the molecular layer59,60 and/or extend novel basal dendrites into the hilus along with mossy fiber cells. The progression of this sprouting is an important consideration because most of the new synapses formed through sprouting take weeks to form, if any form at all.

Excitatory granule cells are not the only neurons that go through morphological changes during epileptic conditions. There is ample evidence to suggest that somatostatin expressing interneurons also increase in soma size,44 sprout axons collaterals39,61 and possibly even sprout dendrites.62,63 These changes in the somatostatin expressing interneuron population are not surprising because they appear to sprout axons in other degenerative neurological conditions as well.64 The somatostain expressing interneurons that sprout new axon collaterals only extend within the range of their normal axonal span, not further along into the septotemporal axis of the hippocampus.65,66,67 However, while somatostain interneurons sprout, PV-IR interneurons tend to prune and shrink during epileptic conditions.56 With all of these potential changes in neuronal morphology surrounding also the potential for neuron death of various populations, it is prudent to analyze what role this sprouting might play in either alleviating seizures or promoting them.

It is important to note that a vast majority of this sprouting takes place inside the hippocampus proper, little sprouting has been identified in the subiculum or the EC.2 However, while neurons in the subiculum tend not to sprout, other neuron populations, most notably pyramidal neurons in the CA1 region can sprout into the subiculum.68 The sprouting is thought to occur as a reactive response to the loss of post-synaptic targets, either through dendrite retraction or neuronal death.69,70 The mechanism which governs sprouting and soma growth/retraction is unknown, but due to this perceived reactive response to lost post-synaptic targets, there is reason to believe that neurotrophins are involved, most notably Brain Derived Neurotrophin Factor (BDNF). The idea is that as the concentration of neurotrophins increase they trigger a concentration dependent response in the neuron that suggests the need for expanded neuronal coverage and the resources to accommodate the expansion. Basically if the axons and/or dendrites grow longer through sprouting, then a larger somata will also be required to maintain a similar level of neuronal efficiency.

The initial intuition when thinking about sprouting, especially interneuron, is that it is a defense mechanistic response to maintain a properly functioning excitatory-inhibitory neuronal network. The generation of a larger target volume through larger somata would increase the probability for perisomatic inhibition from PV-IR interneurons and appropriate somatostatin expressing interneurons. Such a response could attempt to compensate for lost pre-synaptic inputs as well. Also the increased axonal range could form new synapses to compensate for neuronal death or axonal regression due to epileptic conditions. One could even argue that increased granule axon length may stimulate more interneuron-based action potentials in an attempt to increase total inhibitory input into the network.

Unfortunately while all of the above benefits for sprouting appear reasonable, granule/mossy fiber sprouting could also create a significant problem with the maintenance of a properly functioning brain increasing the probability for the development of epilepsy. Normally the dentate gyrus is only able to fire action potentials in a range of (0.1 – 1 Hz)71, which is rather meek. Even when the dentate gyrus creates the rare burst pattern, it remains isolated to the dentate gyrus due to the action of basket cells and the limited interconnections between granule cells.72 This neutralization behavior is in contrast to the pyramidal neurons in the CA3 region which generate frequent burst pattern activity.73 However, at the beginning of epileptic development granule cells begin to die, which stimulate axonal and dendritic sprouting among surviving granule cells in the dentate gyrus. Unfortunately this sprouting facilitates a higher probability of burst pattern firing between granule cells. Add in the loss of inhibitory input, regardless of the reason, and the probability dramatically increases for synchronization and a radically higher excitatory output from the dentate gyrus to the CA1 and CA3 regions, which could generate an epileptic seizure.74,75

At one point in time zinc was considered a molecule of significant interest in trying to explain the cause of epilepsy. The reason for the interest was two-fold. First, Timm’s sulfide-silver staining seemed to demonstrate that zinc was a principle element in the movement, perhaps playing the role of a molecular signal, of sprouting mossy fiber terminals into the outer molecular layer where they are not normally located.68 However, because the method in which zinc may influence any migratory behavior is unknown, it is unclear whether zinc is genuinely influencing migration or is just along for the ride because of its normal physiological location in mossy fiber pre-synaptic cells.

Second, zinc seems to have the ability to reduce the efficiency of GABAA receptors by binding as a form of antagonist.75 The importance of this interaction is that by reducing GABAA binding efficiency for GABA one would expect to reduce the frequency, rate of rise, decay time and amplitude of miniature inhibitory postsynaptic currents (mIPSCs), a result that has been demonstrated.76 However, the efficiency of zinc binding to GABAA is predicated on the presence of epileptic conditions77,78 because zinc effectiveness is dramatically reduced in normal functioning brains. Thus, the influence of zinc seems dependent on the rate of GABAA subunit shift or mutation occurring from epileptic conditions.75 This GABAA subunit requirement seems to significantly limit the role that zinc plays, even in the epileptic brain, at limiting GABA-derived inhibitory potential. The general non-existent influence in normal brains also significantly reduces the probability that zinc initiates epilepsy in any way. Also to interact with GABAA zinc has to travel through an extracellular matrix with a number of free polyvalent anions, which bind zinc making it incompatible with GABAA receptors.75 Therefore, even though there is probably enough zinc available to influence GABAA, the probability that it actually does is rather small. This probability also extends largely to the influence zinc has on NMDA receptors on glutaminergic neurons. Overall regardless of the influence of zinc unless the patient is extreme loading or extremely deficient, zinc probably plays a small role in epileptic derived seizures.

Recall from above that some mossy fiber sprouting results in the formation of novel mossy fiber collaterals, some of which aid synchronization within the dentate gyrus through the development of more functional synapses. Due to the uniqueness of these connections, this mossy fiber dendrite connection to other dentate granule cells is a connectivity that does not appear in normal animals,79 it is possible that these connections are the most critical to fostering synchronized firing in the dentate gyrus. As a result of their excitatory-excitatory connection these novel collaterals seem to act as a recurrent excitatory feedback loop creating a higher likelihood from synchrony and an increased amplitude of excitatory output.

Despite all of the sprouting from interneurons, which some argue can lead to effective replacement of the lost inhibition from dead or dying interneurons, the very nature of epilepsy underlies a reduction of inhibition in proportion to excitation. So it is highly probable that there are other changes in the inhibitory mechanism beyond simple interneuron death that limits the effectiveness of any interneuron sprouting. However, to best understand those changes the very inhibitory network itself must be understood at a basic level.

Recall that GABA is the chief inhibitory neurotransmitter in the brain, but how does it inhibit? GABA can be released through two primary processes from a GABAergic neuron after GABA is transported into a synaptic vesicle at the end of the axon (known as the synaptic bouton). First, there is the process of spontaneous release or a miniature inhibitory post-synaptic potential (mIPSP) where a single vesicle spontaneously fuses and releases its transmitter content (considered one quanta) into the synaptic cleft without the aid of an electrical or chemical trigger. Second, neurotransmitter release is driven through an action potential which dramatically increases the probability of a large quantity of vesicle fusion resulting in a much larger amount of neurotransmitter released into the synaptic cleft. The term readily releasable pool (RRP) is used to describe the general number of synaptic vesicles that could fuse and release neurotransmitter at any given time.

The number of vesicles involved in fusion is not the entire story, each vesicle has a given average concentration of neurotransmitters. Under normal psychological conditions almost all vesicles should have the same concentration of neurotransmitter per vesicle within a given neuron, but under abnormal psychological conditions vesicles may not be filled to the same capacity, which will change the amount of neurotransmitter in the synaptic cleft. So two potential issues in reduced inhibition may be a small number of vesicles that are available for fusion and neurotransmitter release or a smaller quanta of neurotransmitter per vesicle.

PV-IR neuron death, especially in the dentate gyrus, is a controversial issue. The reason being was the depth and placement of their synaptic connections on granule cells. In addition to the placement of these connections, inhibition in the dentate gyrus is also thought to be driven through large quantal sizes and large release probabilities.80 With that said, it is reasonable to believe that a drop in any one of those elements: neurons, quantal size or release probability would reduce inhibition in the dentate gyrus or anywhere else in the hippocampus for that matter.

Even empirical evidence is somewhat controversial regarding the change in inhibition in epileptic patients vs. controls where some have no change76,81,82 or even increasing inhibition83 vs. others that find lower inhibition rates.44,84 Although these results may seem contradictory, there is a possible explanation that could justify them. The first place to start is looking at the excitatory input into the inhibitory elements of the dentate gyrus and the CA regions.

Excitatory inputs defined as evoked mEPSCs are typically measured under four different parameters, average decay time, amplitude, rise time and frequency. Most of the studies concerning mEPSCs and mIPSCs are conducted in animal models (rats and mice). After SE the average decay time, amplitude and rise time for mEPSCs in the dentate gyrus in epileptic rats is generally the same as in control mice,21,44 but mEPSC frequencies are significantly reduced, up to around 50% despite the appearance of mossy fiber sprouting with no significant change in dendritic length in basket cells.44 The most probable explanation for the drop is the death of GluR2 expressing cells in the dentate gyrus (which includes mossy fiber cells).8,9 However, a potentially conflicting piece of evidence is that basket cell dendritic length does not appear to statistically decrease in epileptic patients (after SE).21,65 Any static nature for dendritic length seems counterintuitive. If basket cells are not receiving input or a significantly reduced input then there should be some change in length, either longer in attempt to maintain proper firing rates or shorter as a precursor to neuronal pruning due to a lack of use. Note that while there is mEPSC frequency change in the dentate gyrus, there is also a 50% reduction in mEPSC frequency in the lacunosum moleculare (LM), but no significant mEPSC change in the stratum oriens or the stratum radiatum areas of the CA1 region.7

Some argue a reduction in excitatory input as a result of short-term depression due to a disconnection of basket cells from their afferents85, but if short-term depression were a cause one would expect a significant increase in excitatory input over controls due to increased firing and a steady drop as depression takes over. A mixture of cell death and cell depression could reduce the ability to differentiate between each outcome, thus a labeling means such as Fluro-B Jade may have to be used to track between the two. It is unclear such an observation has been made. Also there is little information regarding whether this potential short-term depression becomes long-term depression or the depressed neuron dies instead.

The death of excitatory neurons in the dentate gyrus may be one explanation for any result that reports a reduced level of inhibition, but it is not the entire story. It has also been reported that in rat models of TLE mIPSCs frequency in granule cells (remember the release is coming from the pre-synaptic basket cell, but is being recorded in the post-synaptic granule cell) drop 35-70% versus control.44,54,86 One suggestion for the decrease in mIPSCs frequency is the death of basket cells (fewer basket cells means lower probability for an mIPSC (i.e. lower frequency)); however, such a suggestion re-ignites the question of interneuron death in the dentate gyrus. Although interneuron death is rather small early into SE, suppose if PV-IR cells are indeed dying then their death could explain such a considerable drop because of the extensive synaptic connections that they have with the granule cells in the dentate gyrus. Basically although the total number of interneurons lost is only a small percentage of the total available, if those that are lost are responsible for more inhibition proportional to the other GABAergic interneuron cell types then the death of these cells would disrupt inhibition of the glutaminergic cells in the dentate gyrus at a greater level than a similar numerical loss of another cell type. However, assume for the moment that the level of interneuron death is not sufficient to account for this result, what else could cause the mIPSC drop?

One explanation could be a reduced RRP, if there are fewer vesicles that are able to fuse at any given time then there is a lower probability that any vesicles would fuse and release neurotransmitter into the cleft. This suggestion is backed up by an increase in uIPSC failure rate to 2.3 times in epileptic rats over control rats.21 Also it has been reported that the average number of vesicles in the RRP for rat controls is 6.421,80,87 whereas epileptic rats have 4.3 vesicles in the RRP.80 While the implication that a reduction in RRP results in less inhibitory input is not surprising the real question is how does this happen? There could be two competing forces, an increase in GAD67 synthesis could increase the demand for synaptic vesicles in the RRP, thus increasing synthesis and/or recycling, but a lack of excitatory input from the dentate gyrus could also reduce the necessity of vesicle synthesis due to a reduced probability of GABA release or there could be a third currently unidentified factor.

As noted above numerous reports identify a significant increase in GAD67 mRNA expression in surviving interneurons under epileptic conditions,88,89 regardless of the extent of cell death or specific hippocampal region.90 GAD65 mRNA expression has also been shown to increase in the subiculum2 and it seems reasonable to assume an increase in expression in other portions of the hippocampus in conjunction with the GAD67 mRNA expression increase. Increases in GAD mRNA expression make sense because a reduction in interneuron inhibitory network ability would drive surviving interneurons to increase GABA release capacity through increasing synthesis rate. However, as mentioned above, the one question with this explanation is if increasing GABA release capacity is important why is the number of vesicles in the RRP reduced?

Currently from the literature it is unclear whether RRPs are smaller because of fewer synaptic contacts, but without any pre-synaptic axonal GABAergic or post-synaptic dendritic glutaminergic regression fewer synaptic contacts would probably not reduce RRP. It would seem to make sense that the retraction would be a necessary signaling step to inform the pre-synaptic neuron to synthesize/recycle fewer vesicles. So while elimination of synaptic targets makes sense for a reduction in overall RRP, without any form of regression it is unclear how the pre-synaptic neuron would know to reduce resource devotion to RRP. One possible means would be the increase/decrease in some secondary messenger, but what it could be remains unclear.

With the introduction of the loss of uIPSCs and mIPSC/mEPSC frequency, there are questions regarding the efficiency of axonal sprouting and whether or not these new axons are actually integrated into the network to the point where they have significant influence on firing patterns. The functionality of these newer synapses is in question as although uIPSC generation create similar patterns of amplitude, charge density, decay time and rise-times between epileptic and control, they could be less reliable than older synapses recalling that the average failure rate for uIPSC generation from these newer synapses is over two times larger than older intact synapses in controls.2,39 Therefore, even though there is a higher probability for a visual or morphological synaptic connection between these particular interneurons and granule cells in epileptic patients, the probability that there is not a psychologically viable connection is also higher. The uIPSCs that are generated in epileptic rats do not appear to be statistically larger than those found in non-epileptic rats (most tests show statistically insignificant lower amplitudes), which suggests that the sprouting does contact a wide variety of newly targeted neurons instead of a small cluster of previously targeted or existing target neurons.

Another way to possibly explore how the RRP or quantal size may change is to look at vesicular GABA transporter (VGAT). The GABA transit system after synthesis (GAD65 and GAD67) involves three other GABA-specific proteins, VGAT, GABA transporter (GAT) and GABA-transanimase (GABA-T). If one would attempt to attribute the lack of mIPSCs in epileptic patents over controls due to a lack of available GABA despite increases in GAD67 expression VGAT would deserve focus.

VGAT is the protein responsible for loading GABA into synaptic vesicles. It would be possible to anticipate a reduction in VGAT expression if there was a reduction in the total number of vesicles in the RRP. A reduction in VGAT expression may actually reduce the overall quantal size. However, despite the possibility for a reduced quantal size that does not appear to be the case. Epileptic rats appear to have an increased quantal size of approximately 1.3-1.5 times control levels at basket neurons to granule neurons synapses.21,91,92 Associate the increased quantal size with an increased number of GABAA receptors per somatic synapse91 and at times one could see an increase in total amplitude of mIPSCs in granule cells under the right conditions.92 Therefore, the answer to the increased/decreased inhibition question seems to be a matter of timing. Basically the total amount of inhibition per trigger is larger (the larger quantal size), but the probability of a trigger is less likely in epileptic patients. Since the frequency of release seems more important than the individual amplitude, there appears to be a overall reduction in inhibition of the entire hippocampal network.

One potentially exotic explanation for the lack of efficient connections for the newer epileptic may be from the limited concentration of GABA itself. During development various types of neuron migrate from their place of origin to their final operating location in the brain. GABA is one of the molecules required for the successful migration of these neurons during development.93,94 What if the lower than normal GABA concentrations in the extracellular matrix due to fewer spontaneous releases and non-fetal conditions do not provide the appropriate molecular cues to facilitate proper axonal migration, thus creating an unreliable synapse? The lingering question with the above idea seems to be whether or not the role of GABA as the chief inhibitory neurotransmitter interferes with this sprouting because GABA plays an excitatory role during development when most of the migration occurs.

Another issue that would most definitely influence inhibitory action in the hippocampus is a change in the probability that a given glutaminergic neuron would express a specific type of subunit on a GABA receptor (GABAA and GABAC). Comparing viable GABAA receptors between control and epileptic animal models is another means to determine extent of cell death, a process that offers further support of significant glutaminergic neuron death in the dentate gyrus, CA1 and CA3 regions.77,95 It is not surprising that given the extent of damage to the hippocampus proper that surviving glutaminergic neurons could experience an increase in the number of GABAA receptors96,97 and even a change in the type of GABAA receptor that they express.95 The most common changes occur after SE and tend to increase the number of alpha1, alpha2, beta2, beta3, gamma2 subunits (in soma and apical dendrites) in the dentate gyrus and decrease the number of alpha1 in the CA1, CA2 and CA3 regions.95 Overall these changes tend to reduce the effectiveness of GABAA when bound with GABA at creating inhibitory post-synaptic potentials.

Despite these conclusions there will still be questions regarding whether or not the granule cells in the dentate gyrus experience an increase or decrease in inhibitory activity. Finding an answer to this question is important because excess excitation of granule cells tend to cause neuronal cell death in the CA1 and CA3 regions of the hippocampus. If inhibition among granule cells increases then it would be difficult to conclude that normal neuronal discharge from granule cells is responsible for neuronal injury or death through the general neuronal interaction pathways, unless of course the inhibition only targeted a certain granule population. If inhibition decreases then it is more probable that normal neuronal discharge from granules cells is a significant component in neuronal death over a more exotic mechanism. Overall it seems probable that although the surviving granule cells try to compensate for the interneurons that are lost through sprouting (trying to increase surviving inhibitory firing rates), they cannot do enough. Interneuronal sprouting failure to form consistent firing synapses seems to help explain why an increase in GAD expression and GABAA expression does not quell seizures.

The question of excitation/inhibition in the dentate gyrus is important, but not the entire story when it comes to seizure onset. Reduced excitatory input to basket cells appears insufficient to cause epilepsy by itself because spontaneous seizures were not observed in mice with reduced AMPA-mediated excitatory synaptic input to parvalbumin-positive basket cells.98 Therefore, while seizure activity may originate in the dentate gyrus the evolution to a full seizure requires other factors. There is an argument that prolonged action potential discharges are also transmitted to distant sites in the brain, which may trigger trains of action potentials in neurons that project back to the neurons in the seizure focus (back-propagation).3 The back-propagation from other regions like the neocortex and the subiculum could provide the necessary evolutionary push for small excess excitatory inputs to expand into potential seizures.

The subiculum is one of the primary output centers from neuronal connections in the hippocampus proper and provides the primary input to the parahippocampal region and EC. Previous studies has demonstrated that spontaneous synchronous firing activity can originate in the subiculum without any input from the CA1 or CA3 regions.99,100 Interestingly in hippocampal slices from epileptic patients which include the subiculum, synchronous interictal activity can be reduced by both glutamate and GABA receptor antagonists.101 The reduction through use of a glutamate receptor antagonist makes sense, but the use of a GABA receptor antagonist does not make sense intuitively, that is unless one remembers that GABA is not always inhibitory.

As previously mentioned, in the mature brain GABA is the principle inhibitory neurotransmitter in the brain largely governing the influx and efflux of chloride ions in relation to neurotransmitter influence. Normally when GABA migrates across the synaptic cleft and binds an appropriate receptor on the post-synaptic neuron it opens a ligand-driven ion channel, which is permeable to chloride. Under normal physiological conditions the chloride concentration inside the neuron is lower than the average chloride concentration outside of the neuron due to the resting electrochemical gradient created by K+Cl- transporters, most notably KCC2.102,103

With a lower internal chloride concentration, chloride ions outside of the cell migrate into the cell along an electrochemical gradient until either the channel closes or a dynamic equilibrium is attained. An increasing chloride concentration inside the cell increases the probability of hyperpolarization, which reduces the probability that the neuron will depolarize firing an action potential. This influx of chloride ions is how GABA typically functions as an inhibitory neuron.

Although GABA is typically inhibitory, at times during neuronal development, under certain pathological conditions and in very specific locations in the mature adult brain GABA can mimic the action of glutamate and depolarize a cell. The ability of GABA to depolarize a neuron is dependent on creating a chloride concentration gradient where there is more chloride inside of the cell than there is chloride outside the cell. The excess chloride in the neuron shifts the equilibrium potential for chloride to a value greater than the normal resting membrane potential (i.e. less negative because the resting membrane potential is about –70 mV). Under these conditions when the GABA-driven chloride channels are opened, chloride will flow out of the cell instead of into the cell, removing the negatively charged chloride ions from the cell increasing the probability of depolarization. This specific chloride concentration gradient is commonly established by a greater expression of NKCC transporters (most notably NKCC1) over KCC2 transporters.102,103 So, a reduction in seizure activity from the application of a GABA antagonist implies that some of the pyramidal neurons in the subiculum depolarize when GABA binds instead of hyperpolarizing.

In the subiculum the behavior of and relationship between regular-spiking neurons, fast-spiking neurons and burst-spiking neurons is important to consider regarding the role of the subiculum in epilepsy. The onset of epiletiform activity appears to originate with the firing of a population of burst-spiking pyramidal cells, behavior that is favored in the subiculum over regions in the hippocampus proper due to the abundance of burst-spiking pyramidal cells in the subiculum.104 The type of burst firing, dependent on intrinsic and synaptic conductances, can develop in one of three ways: 1) desynchronized firing; 2) focal firing; 3) widespread firing;

Clearly burst-spiking neurons firing in a desynchronized pattern are the least likely to result in seizure activity because there is no real coordination between the three different neuron populations, just random fast-spiking and burst-spiking neurons firing.104 Focal firing is characterized by burst neurons firing before paroxysmal field activity occurs with regular-spiking and fast-spiking neurons firing after paroxysmal field activity. 104 Usually during focal firing the fast-spiking interneurons are able to quell any seizure activity by feeding back onto the burst-spiking neurons before full synchronization can be achieved. Widespread firing is similar to focal firing with the exception that instead of firing after paroxysmal field activity is attained, fast-spiking neurons can be driven to fire before field activity begins. 104 Overall the type of firing by burst-spiking neurons in the subiculum heavily influence seizure potential in epilepsy.

Based on this sequence of firing, the burst-spiking neurons that fire first to begin formation of field activity are known as leader neurons and the neurons that fire when paroxysmal activity has already started are known as follower neurons. The defining characteristics that separate leaders and followers seem to be a much larger number of inputs on the distal dendrites in leaders in addition to a lower firing threshold.104,105 Also the leader-follower system in the subiculum generates a level of network connectivity that eases synchronization making seizure activity more likely.105,106

One of the biggest issues in subiculum firing appears to be the inelasticity of the firing order. Although burst-spiking cells are not always the leaders, they are commonly the leaders and when they are followers activity proceeds rather predictability.101 This predictability creates little flexibility for the subiculum to attempt to compensate for any excitatory or inhibitory changes that may occur. In fact despite the question of neuron death, axonal sprouting is not required in the subiculum largely because there isn’t very much, to generate excess excitatory activity and seizure onset, unlike in the dentate gyrus.100,107

While there may not be intra-axonal sprouting in the subiculum, there can be CA1 pyramidal axon intrusion. In a normal rat hippocampus, CA1 neurons project to the subiculum topographically lamellarly.67 However, in epileptic rats, evidence suggests that CA1 neurons extend over a wider range, expanding beyond the normal lamellar boundaries.68 Also CA1 pyramidal neurons have a tendency to become more prone to bursting after a SE episode.108 It is reasonable to suggest that this excitatory expansion can increase amplification and synchronization in the subiculum. In fact the burst firing from the CA1 region after status epilepticus may also induce an increased level of burst firing in subicular neurons (a seven-fold increase) dramatically increasing the probability of recurrent seizure activity.109

There is reason to believe that in an undamaged brain the probability of a seizure is most prevalent during the neonatal and infant period.110,111 Although the reason for this increased seizure potential could vary between neuronal electrotonic coupling, low efficiency potassium buffering or something else, the best explanation could be the larger percentage of GABA depolarizing pyramidal cells in the developing brain over the developed brain. For example take the subiculum, if during development certain populations of burst-spiking neurons were not inhibited by fast-spiking GABAergic interneurons, but further depolarized such a response would increase the probability for seizure development. The reason all infants do not suffer from seizures is that there does not appear to be a specific population of neurons in the subiculum that begin widespread firing, instead widespread firing can originate from different neurons at different times.68,104 Basically seizure development in children could be simple bad luck more than anything else as only a specific burst-spiking neuron group could generate a seizure and other burst-spiking neuron groups could not.

Therefore, based on the above information, the following hypothesis for the development of TLE in young children can be suggested: during the development of the brain GABA is primarily depolarizing due to expression patterns of certain passive chloride transporters. This reversal seems especially prominent in the subiculum making very young children more susceptible to seizures than adults. In the event of a seizure the excitatory input would move from the subiculum into the EC, from the EC to dentate gyrus and CA3 region then to the CA1 region and finally back to the subiculum. Enough activity at a high enough amplitude could generate mossy fiber and interneuron damage in the dentate gyrus which would foster further seizure activity or full-blown epilepsy even after maturation of most subiculum GABAergic depolarizing interneuron into hyperpolarization.

Children that ‘grow’ out of epilepsy could be those that fall into this scenario. Their probability for developing seizures is high when very young, but the frequency or severity of any seizure activity is not enough that it causes sufficient damage elsewhere in the hippocampus to facilitate a second origin condition for future seizure activity. Thus, when the vast majority of neurons in the brain that began as GABA depolarizing evolve to GABA hyperpolarizing, the probability of continuing seizure activity is almost completely eliminated. Overall it seems that most of this seizure activity in young children that could cause the damage that gives rise to epilepsy would be SPS in nature, thus difficult to detect because the absence of initial neuron damage reduces the probability of seizures expanding beyond SPS.

The above theory may offer a possible explanation for idiopathic cases of epilepsy, but would only account for the development of epilepsy in young children not in adults. The most probable explanation for adults is some form of injury or insult damaging the hippocampus, which could start a chain reaction leading to the formation of epilepsy. Now it is a little far-fetched to see how a bump on the head could evolve into epilepsy, but the following explanation may be a possible pathway.

The first element to consider is the vulnerability of neurons in the subiculum. Early studies concluded that neuron loss in the subiculum was rare.112,113 However, more recent studies have demonstrated that there is probably more neuron death, especially interneuron death, in the subiculum than previously thought.2,68,114 There are two reasons interneuron damage or death in the subiculum is important: 1. the loss of fast-spiking inhibition; 2. the up-regulation and release of BDNF; excess excitation stemming from the first reason is rather obvious, less inhibition will lead to excess excitation. Excess excitation stemming from the second reason is a little trickier. BDNF is a neurotrophin that reduces the probability of cell death, facilitate faster neuronal sprouting, growth and repair and is commonly released by cells when under distress. Continuous seizures would create scenarios of excitotoxicity and glucose/oxygen related distress, thus an increase in BDNF expression would make logical sense and has been experimentally demonstrated.115

Normally the release of BDNF would be viewed as a positive because an increased concentration would increase survival rate; however, BDNF has also been shown to up-regulate NKCC transporters and down-regulate KCC2 transporters.116,117,118,119 Recalling the depolarizing/hyperpolarizing characteristics of GABA, the more NKCC transporters a neuron has the larger its internal chloride concentration, which will result in the outflow of chloride instead of inflow upon GABA binding increasing the probability for depolarization. The interesting thing about this BDNF behavior is that during neuronal development in young children BDNF actually increases KCC2 expression, but after development the larger concentration of trkB receptor seems to reverse this expression behavior where BDNF actually reduces KCC2 expression in favor of NKCC.117,118 The relationship between BDNF and KCC transporters in very young children is another neuroprotective measure that may be used by the brain naturally to prevent the development of epilepsy. Regarding the reversal of the BDNF influence, one possible long-shot therapeutic strategy for treating epilepsy may be to administer a weak trkB receptor antagonist to help neutralize the effect of BDNF related up-regulation of NKCC transporters. The antagonist needs to be weak because trkB serves a critical role in other processes.

It is also interesting to note that BDNF has been implicated in release probability and RRP size at GABAergic synapses.120 This augmentation ability may neutralize the RRP loss that some suspect occurs in epileptic patients, but if BDNF is also increasing the probability that an excitatory neuron becomes GABA depolarizing over GABA hyperpolarizing another aspect of BDNF that should be beneficial would turn into detrimental in seizure progression as increasing GABA concentration in the synaptic cleft would increase the probability of GABA-based depolarization.

Therefore, with these changes caused by BDNF the attempts of subiculum interneurons to survive and maintain input/output balance in the subiculum may actually worsen the problem by briefly converting subicular pyramidal neurons from GABA hyperpolarizing to GABA depolarizing thereby increasing amplitude and duration of the excitatory impulse. With approximately 20-22% of subicular pyramidal neurons being GABA depolarizing,68 in the normal adult brain any increase in GABA depolarizing pyramidal neurons through an excess BDNF concentration could very well increase the probability of seizure onset and progression.

In adult epilepsy the subiculum can be viewed as a form of echo chamber. Under normal conditions the initial signal (shout) by the dentate gyrus is not loud (large) enough to trigger any significant amplification or feedback. In an epileptic adult brain there is a lack of necessary inhibitory control in some of the shouts, which breaks the preverbal ‘echo threshold’ in the subiculum creating a rapid and significant amplification leading to a seizure and possible excitotoxicity damage if SE occurs, which further increases the probability of future seizures.

One lingering question is how do these reversal GABA cells in the subiculum develop beyond a young age? There are a number of possibilities: first, their existence is the result of a genetic mutation which may make some individuals more susceptible to epileptic seizures than others, which may make sense because there is a small genetic component to epilepsy; second, their existence is natural where a large percentage of those reversal neurons are generally conserved and all humans have them; third, their generation is the result of neuronal damage to the subiculum and the lack of a particular mophogen or other signal when the brain attempts to repair the damaged cell; if the methodology behind how this reversal occurs could be identified, if it is not just a conserved region of the brain, then perhaps these cells could have a more targeted focus for treatment of epilepsy. Unfortunately if the second option is correct there is no definitive information regarding how essential these default neurons are to a proper functioning hippocampus, so thus therapeutically targeting these neurons may not be advisable regardless of developmental methodology. Overall it appears that epilepsy in general, whether its origins are from a developmental level or a later brain injury depend on the presence of subicular pyramidal neurons that are GABA depolarizing.

One interesting question regarding the role in generation of GABA depolarizing neurons as a significant driving force in the progression of epilepsy is why most drug therapies for epilepsy that utilize factors which increase GABA concentration or effectiveness in the synaptic cleft work? If depolarizing GABA neurons played a significant role in seizure generation, one would suspect that these AEMs would be a failure instead of a minor success. A possible solution to this question is if depolarizing GABA neurons enhance seizure activity through excess excitation or synchronization, but have nothing to do with the initial seizure origin. For example the influence of depolarizing GABA neurons in the subiculum can be viewed as domino number 4 in the progression to a seizure as it knocks over dominos 5-9 at the same time, but the AEMs attempt to augment GABA to stop seizure progression at domino number 1 or 2 (dentate gyrus or CA3). If domino number 1 is stopped, the effect of domino number 4 is meaningless. Seeing that most of the depolarizing GABA neurons reside in the subiculum and scant numbers appear in the dentate gyrus, CA1 or CA3 regions, this explanation could hold some weight.

Gap junctions appear to be important with relation to electrical coupling and gamma level synchronization. It is possible that significant amplification of excitotoxicity potential occurs through gap junction synchronization. Unfortunately there is little that can be done with this information because applying any agent that could block gap junction and reduce the probability of this synchronization would have to be highly localized within the damaged region. If the agent could not be isolated to a localized region then the resultant collateral damage to healthy neurons could be worse than the epilepsy. Additionally it is unclear how using a gap junction blocking agent would affect epileptic seizures because it is unclear how much influence gap junction synchronization has in initiating and maintaining a seizure. Despite this uncertainty it is reasonable to assume that synchronization disruption would reduce the severity of a seizure.

Ketogenic diets have also been shown to have a level of success in reducing the onset of seizure activity in epileptic patients.121,122,123 Interestingly enough while the mechanism behind the success of the ketogenic diet is currently unknown, a rational hypothesis can be suggested when focusing on the hypothalamus response to food intake. Not surprisingly specific food intake can be biologically conditioned based on the release of certain molecules in the brain and visa-versa. For example excess release of norepinephrine into the paraventricular nucleus increases the probability for consumption of carbohydrates over proteins and fats.3 Increasing carbohydrate consumption will increase norepinephrine release. A ketogenic diet is very high in fats and low in carbohydrates. Among other responses one of the biological responses to the consumption of fats is the release of the peptide galanin. Galanin has demonstrated anti-convulsive properties124,125 through reducing the concentration of glutamate released via pre-synaptic inhibition,126 so enhanced release of galanin in the brain could reduce the frequency and severity of seizures, which is what is seen in a number of patients on a ketogenic diet. Note that the important issue here is the release of galanin, not mRNA expression as galanin has almost no anti-convulsent influence unless precipitated from the cell. However, whether or not an increase in release actually happens is unknown.

One reason for the effectiveness of galanin may be location. The dentate gyrus under normal conditions has the highest density of galanin-immunoreactive fibers and it makes sense that galanin concentration would increase periodically in an epileptic brain.125 Unfortunately the reason galanin may not play as important of a role as it should is that the total available concentration is quickly exhausted under seizure conditions.126 Any treatment with a synthetic non-peptide version of galanin (which is necessary because peptides have difficulties crossing the blood-brain barrier) will need to focus on the difference between the two galanin receptors, GalR1 and GalR2. Interaction with GalR1 reduces the probability of seizure onset, but seems to do little once the seizure as begun and interaction with GalR2 reduces the length of a seizure, but does little to prevent seizure onset.125,127,128 A combination of a ketogenic diet and a synthetic galanin which binds to GalR2 may be more effective at quelling seizure activity for most patients than either alone.

Based on the above information it may be possible to develop an epilepsy ‘vaccine’. Although the use of the term ‘vaccine’ is not completely accurate, the general concept is the same. The injection of an appropriate chemical soon after birth could reduce the influence of the GABA depolarization effect over the GABA hyperpolarization effect, which could reduce the probability that the given child develops epilepsy. Two early candidate for such a ‘vaccine’ could be additional BDNF or IGF-1. IGF-1 is a candidate because there is evidence that IGF-1 hastens post-translational modifications of KCC2 increasing its overall activity level.129 However, the roles of both IGF-1 and BDNF in early development would need to be further studies as well as the role of the GABA depolarizing glutaminergic neurons in the subiculum to ensure that such a treatment would do more good than harm.

Another potential treatment option is addressing the possible ‘unique’ novel basal dendrites that sprout from the mossy fiber cells in the dentate gyrus. It stands to reason that if these dendrites typically do not appear in a normal brain then their generation is facilitated by a novel molecular signal or novel signal sequence facilitated by existing molecules. If such a signal exists and it can be identified then it may be possible to specifically target those novel dendrites and either preventing their sprouting in the first place or terminate them with little overall damage to the rest of the mossy fiber and/or granule cell network, knocking out the synchronization required for seizure generation.

Although epilepsy is rather muted when it comes to public recognition, it prevalence is still significant throughout the world with TLE being the most probable. There should be two general attack strategies for dealing with TLE, first prevent its occurrence and second therapeutically treat its symptoms. Prevention appears viable if one could devise some means of reducing the probability of action potential firing in the subiculum during neural development in very young children, which should prevent the development of TLE from propagation of excessive subiculum excitation. Treatment would be improved through more unique targeting mechanisms, such as identifying whether or not the novel dendrites that form in the dentate gyrus are a viable treatment option. Also the combination of form of galanin agonist with a ketogenic diet may offer more pronounced seizure control than either alone. In the future stem cell treatments may provide another level of seizure control through the restoration of lost or non-functional interneurons in all regions of the hippocampus, especially those in the dentate gyrus.
Overall a better understanding of the new methodological discoveries of epilepsy development and maintenance should lead to a higher rate of success for combination therapies such as synthetic galanin and a ketogenic diet or early preventative measures such as injection of some chemical just after birth.


Afferent Neurons: Neurons that carry nerve impulses from sensory receptors to the CNS.

Ammon’s Horn Sclerosis (a.k.a. cornu ammonis (CA)): The most common types of neuropathological damage in temporal lobe epilepsy. Approximately 65% of people suffering from what can be classified as TLE suffer from this type.

Axo-axonic neurons: Neurons that have axons which connection to the axons of other neurons. Also commonly referred to as ‘chandelier cells/neurons’.

Deafferentation: When a neuron does not have at least one operational afferent connection with other neurons;

Ectopic: A displacement or malposition of a cell or an organ;

Efferent Neurons: Neurons that carry nerve impulses from the CNS to sensory receptors.

Fascia Dentata: Earliest stage in the hippocampal pathway and with the hilus makes up the dentate gyrus. Receives input from the perforant path from the entorhinal cortex. Houses tiny granule cells which project mossy fibers that interact with the hilus and CA3 region of the hippocampus.

Gliosis: A proliferation of astrocytes in damaged areas of the CNS.

Granule Cells: A generally tiny and unmyelinated neuron (10 micrometers in diameter). In relation to epilepsy most relevant granule cells are located in the dentate gyrus and have glutamatergic projection axons. The axons project from the basal portions of the granule cells. Granule cells commonly receive excitatory inputs from mossy fibers.

Hippocampal Formation: term used to encompass the hippocampus proper, dentate gyrus and the subiculum.

Hippocampus Proper: term used to encompass the four CA fields.

Ictal: A physiologic state or event such as a seizure, stroke and headache.

Interictal activity: Neural activity in an epileptogenic region in the time between two seizures.

Ischemia: A restriction in blood supply or flow and there by a lack of oxygen, glucose, etc.; typically related to the brain, commonly resulting in cell damage.

Mossy Fibers: A thick bundle of axons projecting from specific granule cells that bridge the dentate gyrus to the stratum lucidum in the CA3 region providing excitatory input to pyramidal cells in that region. They also provide excitatory input to interneurons in the hilar region of the dentate gyrus.

Neuronal Sprouting: Growth of axons and/or dendrites from a neuron that has either been damaged or a neuron neighboring a damaged neuron. The growth is thought to be driven by a psychological response to compensate for the damage.

Orthodromic Impulse: Orthodromic impulses are a fancy name to describe normal axonal impulses that move towards the synaptic buton and away from the soma.

Parahippocampal Region: A region of the hippocampus that includes the entorhinal cortex (EC), the perirhinal cortex (PC) and the posterior parahippocampal cortex.

Paroxysmal Depolarizing Shift (PDS): Commonly viewed as a hallmark of epilepsy. PDS is defined as a calcium mediated depolarization leading to the influx of sodium into the neuron commonly resulting in an action potential. The action potential is later quelled by a hyper-polarization event through either the influx of chloride or the eflux of potassium.

Parvalbumin: A calcium binding albumin protein. In relation to epilepsy, parvalbumin is contained in basket, axo-axonic, bistratified and oriens-lacunosum moleculare (O-LM) GABAergic interneurons in the hippocampus. Each of these cell types are fast-spiking and targets a distinct group of pyramidal cells. PV-expressing interneurons represent approximately 25% of GABA cells in the primate DLPFC.

Perisomatic Inhibition: Inhibition that is applied to the soma of the post-synaptic cell instead of the dendrites.

Phasic Firing: Descriptive measure of neurons that fire in bursts;

Recurrent Inhibition: The reduction of motor neuron discharge through feedback circuits involving axon collaterals which excite interneurons. Such a feedback system is used to prevent repeat firings of motor neurons over initial firings.

Somatostatin: A peptide inhibitory hormone that regulates the endocrine system and affects neurotransmission and cell proliferation through secondary messenger G-proteins. Interactoin with these G-proteins leads to inhibition of secondary messenger hormones.

Status Epilepticus: Repeated generalized seizures without return to full consciousness between seizures with a large probability for permanent brain damage. The seizure activity typically has to occur for more than 30 minutes.

Stratum Oriens: Located in the CA1 region of the hippocampus below the alveus region. Contains a large number of GABAergic interneurons (basket and horizontal trilaminer cells) that project into basal dendrites and soma of pyramidal cells in the hippocampus. Also the basal dendrites of pyramidal neurons are located in this region and receive input from other pyramidal cells in the CA3 and CA2 regions.

Zonula Adherens: Protein complexes that occur at cell-cell junctions in epithelial tissues. Cadherins, β-catenin and α-catenin are the constituents largely responsible for zonula adherens.


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