Chong L. Lee

Spine loss and other dendritic abnormalities in epilepsy

Studies of neurons from human epilepsy tissue and comparable animal models of focal epilepsy have consistently reported a marked decrease in dendritic spine density on hippocampal and neocortical pyramidal cells. Spine loss is often accompanied by focal varicose swellings or beading of dendritic segments. An ongoing excitotoxic injury of dendrites (dendrotoxicity), produced by excessive release of glutamate during seizures, is often assumed to produce these abnormalities. Indeed, application of glutamate receptor agonists to dendrites can produce both spine loss and beading. However, the cellular mechanisms underlying the two processes appear to be different. One recent study suggests NMDA‐induced spine loss is produced by Ca2+‐mediated alterations of the spine cytoskeleton. In contrast, dendritic beading is not dependent on extracellular Ca2+; instead, it appears to be produced by the movement of Na+ and Cl− intracellularly and an obligate movement of water to maintain osmolarity. A decrease in dendritic spine density was recently reported in a model of recurrent focal seizures in early life. Unlike results from other models, dendritic beading was not observed, and other signs of neuronal injury and death were absent. Thus, additional mechanisms to those of excitotoxicity may produce dendritic spine loss in epileptic tissue. A hypothesis is presented that spine loss can be a product of a partial deafferentation of pyramidal cells, resulting from an activity‐dependent pruning of neuronal connectivity induced by recurring seizures. The dendritic abnormalities observed in epilepsy are commonly suggested to be a product and not a cause of epilepsy. However, anatomical remodeling may be accompanied by alterations in molecular expression and targeting of both voltage‐ and ligand‐gated channels in dendrites. It is conceivable that such changes could contribute to the neuronal hyperexcitability of epilepsy. Hippocampus 2000;10:617–625.

Spatial learning deficits without hippocampal neuronal loss in a model of early-onset epilepsy

Studies were undertaken to examine the effects recurrent early-life seizures have on the ability of rats to acquire spatial memories in adulthood. A minute quantity of tetanus toxin was injected unilaterally into the hippocampus on postnatal day 10. Within 48 h, rats developed recurrent seizures that persisted for 1 week. Between postnatal days 57 and 61, rats were trained in a Morris water maze. Toxin-injected rats were markedly deficient in learning this task. While these rats showed gradual improvement in escape latencies over 20 trials, their performance always lagged behind that of controls. Poor performance could not be explained by motor impairments or motivational difficulties since swimming speed was similar for the groups. Only eight of 16 toxin-injected animals showed focal interictal spikes in the hippocampus during electroencephalographic recordings. This suggests that learning deficiencies and chronic epilepsy may be independent products of recurrent early-life seizures. A quantitative analysis of hippocampus revealed a significant decrease in neuronal density in stratum pyramidale of experimental rats. However, the differences were largely explained by a concomitant increase in the area of stratum pyramidale. Studies of glial fibrillary acidic protein expression and spread of horseradish peroxidase-conjugated tetanus toxin in the hippocampus suggest that the dispersion of cell bodies in stratum pyramidale can neither be explained by a reactive gliosis nor the direct action of the toxin itself. Taken together, we suggest that recurrent seizures beginning in early life can lead to a significant deficiency in spatial learning without ongoing hippocampal synchronized network discharging or a substantial loss of hippocampal pyramidal cells.

A new animal model of infantile spasms with unprovoked persistent seizures

Purpose: Infantile spasms is one of the most severe epileptic syndromes of infancy and early childhood. Progress toward understanding the pathophysiology of this disorder and the development of effective therapies has been hindered by the lack of a relevant animal model. We report here the creation of such a model.

Tetanus toxin-induced seizures in infant rats and their effects on hippocampal excitability in adulthood

A new experimental model of developmental epilepsy is reported. Behavioral and EEG features of seizures produced by unilateral intrahippocampal injection of tetanus toxin in postnatal day 9-11 rats, are described. Within 24-72 h of tetanus toxin injection, rat pups developed frequent and often prolonged seizures which included combinations of repetitive wet dog shakes, and wild running-jumping seizures. Intrahippocampal and cortical surface EEG recordings showed that coincident with these behaviors, electrographic seizures occurred not only in the injected hippocampus, but also in the contralateral hippocampus and bilaterally in the neocortex. Analysis of the interictal EEG revealed multiple independent spike foci. One week following tetanus toxin injection, the number of seizures markedly decreased; however, interictal spiking persisted. After injection rats were allowed to mature some were observed to have unprovoked behavioral seizures and/or epileptiform EEG activity. Mature animals were also studied using in vitro slice techniques. Recordings from hippocampal slices demonstrated spontaneous epileptiform burst discharges in the majority of rats which had tetanus toxin induced seizures as infants. These events occurred in area CA3 and consisted of interictal spikes and intracellularly recorded paroxysmal depolarization shifts (PDSs). On rarer occasions, electrographic seizures were recorded. The use of the tetanus toxin model in developing rats may facilitate a better understanding of the unique features of epileptogenesis in the developing brain and the consequences early-life seizures have on brain maturation and the genesis of epileptic conditions in later life.

Recurrent seizures and the molecular maturation of hippocampal and neocortical glutamatergic synapses.

Recurrent seizures in animal models of early-onset epilepsy have been shown to produce deficits in spatial learning and memory. While neuronal loss does not appear to underlie these effects, dendritic spine loss has been shown to occur. In experiments reported here, seizures induced either by tetanus toxin or flurothyl during the second postnatal week were found to reduce the expression of NMDA receptor subunits in both the hippocampus and neocortex. Most experiments focused on alterations in the expression of the NR2A subunit and its associated scaffolding protein, PSD95, since their expression is developmentally regulated. Results suggest that the depression in expression can be delayed by at least 5 days but persists for at least 3–4 weeks. These effects were dependent on the number of seizures experienced, and were not observed when seizures were induced in adult mice. Taken together, the results suggest that recurrent seizures in infancy may interrupt synapse maturation and produce persistent decreases in molecular markers for glutamatergic synapses – particularly components of the NMDA receptor complex implicated in learning and memory.

High frequency EEG activity associated with ictal events in an animal model of infantile spasms.

Purpose:u2002 To describe high frequency (HF) electrographic activity accompanying ictal discharges in the tetrodotoxin (TTX) model of infantile spasms. Previous studies of HF oscillations in humans and animals suggest that they arise at sites of seizure onset. We compared HF oscillations at several cortical sites to determine regional differences.

Neuronal activity and the establishment of normal and epileptic circuits during brain development.

The question we attempted to address in this chapter is: Do brief but recurrent seizures in early life alter the ontogeny of hippocampal networks in ways that produce epileptic circuits? Results from the tetanus toxin model suggest that this is likely the case. Following seizures in Postnatal Weeks 2 and 3, most adult rats have a focal epilepsy that arises from hippocampus. Recordings from hippocampal slices support this conclusion since they demonstrated the occurrence of spontaneous network discharges in normal artificial cerebrospinal fluid. Moreover, when GABA-A receptor-mediated synaptic transmission was suppressed, slices from adult epileptic rats produced prolonged electrographic seizures which are never observed in control rats. This suggests that hyperexcitable recurrent excitatory networks contribute to hippocampal seizures in this model. In light of this, anatomical results from biocytin-filled neurons were surprising. Results suggest that recurrent axon arbors neither sprout additional branches as a result of seizure activity nor maintain their exuberant branching patterns of early life. Thus, excessive connectivity cannot explain seizure generation. Axon arbors either remodel in normal ways or prune additional collaterals as a result of ongoing epileptiform discharging. At the same time that axon arbors remodel, the dendrites of these cells have decreased dendritic spine density, suggesting a partial deafferentation. While a complete understanding of the origins of spine loss requires further investigation, we hypothesize that this loss is a product of a partial deafferentation that occurs due to excessive and abnormal selection of synaptic connections. Network-induced heterosynaptic LTD of noncoincidentally active afferants may be one mechanism that leads to a loss of synapses. Moreover, competition among and selection between individual recurrent excitatory synapses may contribute to spine loss as well. The winners of this competition, the most potent and effective early-formed recurrent excitatory synapses, are likely key contributors to seizure generation in this model and possibly in humans with early-onset temporal lobe epilepsy.

Interictal High Frequency Oscillations in an Animal Model of Infantile Spasms

While infantile spasms is the most common catastrophic epilepsy of infancy and early-childhood, very little is known about the basic mechanisms responsible for this devastating disorder. In experiments reported here, spasms were induced in rats by the chronic infusion of TTX into the neocortex beginning on postnatal days 10-12. Studies of focal epilepsy suggest that high frequency EEG oscillations (HFOs) occur interictally at sites that are most likely responsible for seizure generation. Thus, our goal was to determine if HFOs occurred and where they occurred in cortex in the TTX model. We also undertook multiunit recordings to begin to analyze the basic mechanisms responsible for HFOs. Our results show that HFOs occur most frequently during hypsarrhythmia and NREM sleep and are most prominent contralateral to the TTX infusion site in the homotopic cortex and anterior to this region in frontal cortex. While HFOs were largest and most frequent in these contralateral regions, they were also commonly recorded synchronously across multiple and widely-spaced recordings sites. The amplitude and spatial distribution of interictal HFOs were found to be very similar to the high frequency bursts seen at seizure onset. However, the latter differed from the interictal events in that the high frequency activity was more intense at seizure onset. Microwire recordings showed that neuronal unit firing increased abruptly with the generation of HFOs. A similar increase in neuronal firing occurred at the onset of the ictal events. Taken together, results suggest that neocortical networks are abnormally excitable, particularly contralateral to TTX infusion, and that these abnormalities are not restricted to small areas of cortex. Multiunit firing coincident with HFOs is fully consistent with a neocortical hyperexcitability hypothesis particularly since they both occur at seizure onset.

Vigabatrin therapy implicates neocortical high frequency oscillations in an animal model of infantile spasms.

Abnormal high frequency oscillations (HFOs) in EEG recordings are thought to be reflections of mechanisms responsible for focal seizure generation in the temporal lobe and neocortex. HFOs have also been recorded in patients and animal models of infantile spasms. If HFOs are important contributors to infantile spasms then anticonvulsant drugs that suppress these seizures should decrease the occurrence of HFOs. In experiments reported here, we used long-term video/EEG recordings with digital sampling rates capable of capturing HFOs. We tested the effectiveness of vigabatrin (VGB) in the TTX animal model of infantile spasms. VGB was found to be quite effective in suppressing spasms. In 3 of 5 animals, spasms ceased after a daily two week treatment. In the other 2 rats, spasm frequency dramatically decreased but gradually increased following treatment cessation. In all animals, hypsarrhythmia was abolished by the last treatment day. As VGB suppressed the frequency of spasms, there was a decrease in the intensity of the behavioral spasms and the duration of the ictal EEG event. Analysis showed that there was a burst of high frequency activity at ictal onset, followed by a later burst of HFOs. VGB was found to selectively suppress the late HFOs of ictal complexes. VGB also suppressed abnormal HFOs recorded during the interictal periods. Thus VGB was found to be effective in suppressing both the generation of spasms and hypsarrhythmia in the TTX model. Vigabatrin also appears to preferentially suppress the generation of abnormal HFOs, thus implicating neocortical HFOs in the infantile spasms disease state.

Developmental Neuroplasticity and Epilepsy

Results from numerous experimental and clinical studies suggest that the immature nervous system is unusually susceptible to seizures during periods of postnatal life. In rats and mice, this period is postnatal weeks 2 and 3. Similar to that in human neonates, the first week of life in rodents is marked by poorly synchronized neuronal discharging. However, by postnatal week 2, a dramatic transition occurs, and animals demonstrate a marked increase in epileptogenicity reflected in increases in rates of neocortical focal epileptogenesis, amygdala and hippocampal kindling, and susceptibility to convulsant drugs (1). This critical period of enhanced seizure susceptibility has also been demonstrated in in vitro models, which permit more detailed examination of the underlying processes. For instance, when in vitro slices are taken from the hippocampus of 2and 3-week-old rats and exposed to convulsant drugs, such as GABA,-receptor antagonists, they generate prolonged electrographic seizures. However, under identical recording conditions, slices taken from rats <5 days of age do not produce synchronized events, and slices from adult rats produce only brief interictal discharges (2). A late onset of GABA-mediated synaptic inhibition could conceivably play a role in enhanced seizure susceptibility. However, this seems unlikely, given that GABA,-receptor antagonists are such potent convulsants in early life. On the contrary, GABA,-mediated synaptic inhibition appears to be all important in preventing seizures. Electrophysiologic, pharmacologic, neurochemical, and anatomic results all suggest that excitatory amino acid synaptic transmission is enhanced during postnatal weeks 2 and 3 and could contribute to seizure generation (3). This is particularly true in hippocampal area CA3, where recurrent excitatory synapses between mutually excitatory pyramidal cells play an important role in seizure generation. Computer reconstruction of recurrent axon arbors from single CA3 pyramidal cells have shown that few axonal branches are present before the critical period of seizure susceptibility. However, by postnatal days 9 and 10, an exuberant outgrowth has taken place. Half of these axonal branches are pruned by adulthood. Thus, there is good correlation between developmental remodeling of excitatory connectivity and changes in seizure susceptibility (4). In general, remodeling of neuronal connectivity during brain development is dependent on neuronal activity (5). The correlated or synchronous activation of synapses is thought to lead to their selection and maintenance into adulthood. Inactive synapses or synapses activated out of phase with other more effective inputs to cells are thought to be selected against and pruned. Thus, investigators have questioned what the effects of recurring seizures are on the formation of neuronal networks. To address this question, the tetanus toxin model was first developed (6). A unilateral intrahippocampal injection of tetanus toxin on day 10 produces brief but recurrent seizures that decrease in frequency after 1 week. As adults, these rats are chronically epileptic, as demonstrated by video-EEG recordings and recordings from CA3 pyramidal cells in vitro slices (6,7). Results of slice experiments also suggest that hyperexcitable CA3 recurrent excitatory networks play a key role in seizures in these rats (8). To determine the anatomic substrates for the epileptiform discharges, individual CA3 pyramidal cells were labeled intracellularly with biocytin (9). Several outcomes were possible. Most often, speculation has been that early-life seizures “freeze” developing networks into immature patterns of connectivity. For instance, during hippocampal development, when repeated seizures occur, the synchronous discharging of pyramidal cells might prevent axonal remodeling that normally takes place in area CA3. Thus, the excess in axonal branching that is present in early life could be maintained into adulthood. However, other outcomes are possible. The growth of recurrent axon arbors that take place during week 2 could be promoted by seizure discharges. Sprouting of recurrent excitatory collaterals could produce profuse innervation by local networks. A third outcome would follow from results of studies of the neuromuscu-