- Top of page
- Materials and Methods
Purpose: Studies have suggested that the medial dorsal nucleus of the thalamus plays a role in the behavioral expression of limbic seizures, but it is unclear whether this region is a key component for the primary seizure circuitry or a path for seizure spread from one region to another. This study was undertaken to determine the potential role of this region in limbic seizure activity.
Methods: Adult male rats received kindling stimulation either under urethane anesthesia or while awake. Glutamate or its agonists or the GABA antagonist bicuculline or agonist muscimol were infused into the medial dorsal nucleus. In another series, kindling acquisition was compared among three thalamic sites as well as with the amygdala and hippocampus
Results: Drugs that enhanced excitatory drive or blocked GABA resulted in significant prolongation of electrographic seizure activity compared to saline infused controls. Enhanced GABA activity resulted in a significant reduction of seizure duration. Infusion of the compounds lateral to the medial dorsal nucleus did not affect seizure duration. In the kindling studies the medial dorsal region is the only thalamic nucleus from which hippocampal seizures can be induced, but with an elevated afterdischarge threshold compared to the two limbic sites. However, the seizures generalized more rapidly from the medial dorsal region.
Conclusions: This study demonstrates that the medial dorsal nucleus and other dorsal midline nuclei have a significant role in the primary seizure circuits of limbic seizures as well as in spread of seizure activity to other regions.
Seizures occur in neuronal networks, and there is good evidence that the various components in that network play different roles (Avoli & Gloor, 1982; Lothman et al., 1991; Meeren et al., 2002). Understanding the make up of a seizure network and the role each component plays is important for selecting targets for therapeutic intervention.
Spike and wave seizures of absence epilepsy have provided a strong conceptual basis for the idea of a seizure network with different roles for the separate components. The primary network has been defined as a reciprocal circuit involving the neocortex and thalamic relay nuclei with the thalamic reticular nucleus serving as a key modulator (Avoli & Gloor, 1982; Meeren et al., 2002). In this functional model, the cortex provides the excitatory drive, and the thalamus serves to organize this drive into the spike wave pattern.
The role of the thalamus in other types of epilepsy is understood less well. Recordings from human epilepsies from this region are not performed very often, but there is clear evidence of thalamic involvement in the seizures, although the role of the thalamus in the seizures is not well defined (Guye et al., 2006). Recent human clinical trials have targeted various thalamic nuclei for therapeutic electrical stimulation in attempts to control the epilepsy (Kerrigan et al., 2004; Andrade et al., 2006), with mixed success. However the success or failure of this approach with a limited array of stimulation protocols cannot define the role of this region with its complex connections. Animal studies have also not had overwhelming success (Lado, 2006).
Mesial temporal lobe or limbic epilepsy is a specific form of epilepsy that is associated with anatomical changes in a number of regions including the hippocampus, entorhinal cortex and amygdala (Du et al., 1993; Hudson et al., 1993; Margerison & Corsellis, 1966). Recent evidence suggests that there are anatomic changes in the medial dorsal (MD) region of the thalamus associated with this form of epilepsy (Juhász et al., 1999; Bertram et al., 2001; Natsume et al., 2003), and there are initial suggestions from animal models that it may be involved with seizure activity from the earliest stages (Bertram et al., 2001). There is, however, a recent study that suggests that thalamic involvement is apparent only in the later stages of kindling if the rats are kindled in the amygdala (Blumenfeld et al., 2007).
There have been a number of previous studies (kindling and status epilepticus) that have suggested that the MD region could play a role in seizure spread (Lothman & Collins, 1981; Patel et al, 1988; Hirayasu & Wada, 1992; Cassidy & Gale, 1998; Bertram et al., 1998, 2001). In the present study, we examined whether modulation of synaptic activity in the MD region could influence limbic seizure activity to delineate further the potential role of this area in limbic seizures. We also examined the ability to induce seizures from the thalamus using the kindling model to determine whether this region could be a source for seizure initiation. The overall goal of these experiments was to define the role of the MD region in limbic seizures and to determine whether the seizures could be modified by influencing synaptic activity.
- Top of page
- Materials and Methods
This study demonstrates several points. First, pharmacological manipulation of the midline thalamic region, including the MD and paraventricular nuclei, can modulate seizure activity initiated in the hippocampus. Second, although it is more difficult to stimulate a seizure from the MD region than from the hippocampus or amygdala, seizures stimulated from this area generalize very quickly, suggesting that this thalamic region may play an important role in the spread of the seizures. Third, the limbic seizures can be altered by a variety of receptor agonists and antagonists infused into the dorsal midline region, indicating that limbic seizures are influenced by modulation of synaptic activity in this area. Finally, the late involvement of several lateral thalamic nuclei in seizures induced by limbic stimulation as well as the difficulty in initiating limbic or other seizures by stimulation of these relay nuclei suggest that the midline thalamic region is the one that is specifically involved in the initial stages of limbic seizures. These observations expand and more fully define preliminary observations reported before and clearly indicate that these midline nuclei play a significant role in the circuitry of limbic seizures, including the initiation and spread.
A question may arise with regard to the use of the anesthetized animals as well as the awake animals in this study. Both models have been regularly used in the laboratory for many years to answer specific questions. The advantage of using kindling under urethane is that the seizures don't rapidly involve multiple areas (implying restricted circuit recruitment), are relatively short in duration, can be given very close together, and show only a gradual change in duration over time so that they offer a very stable pattern for intervention. Under anesthesia, the placement of electrodes and maintaining an exact placement is much easier than in awake, chronic animals. Further the multiple electrodes and the acute infusions are much easier to place with a multiple arm stereotactic frame. There are potential disadvantages to using urethane as well. The seizures are more difficult to elicit and are shorter in duration. This observation suggests that the urethane has a seizure suppressive effect that may limit the number of neuronal circuits that are involved. It is not known which circuits could be more susceptible to these suppressive effects, and if key circuits are the primary target of the drug. On the other hand the advantage may be that, if there are fewer circuits involved because of the anesthesia, that it is easier to isolate several. It is because of this issue of anesthesia-induced seizure suppression that the use of awake animals is necessary to corroborate any findings obtained under anesthesia. The awake animals offer the advantage of having full behavioral seizures that involve multiple circuits so that one can get a better overview of the likely interactions and roles of many different sites. The primary disadvantage is that because of the involvement of more brain areas with longer seizures, the seizures may recruit additional areas that create a larger thalamic target zone. The results of the muscimol infusion study in the awake kindled animals suggests that the thalamic targets for the initial seizure initiation of limbic seizures remain relatively restricted.
The circuitry of epilepsy has been of interest for many decades. That seizures spread in the brain in some pattern that may be predicted by the connectivity of one region to another or by the recruitment of adjacent areas has been surmised at least since the first descriptions of Jacksonian march. In this study, we examined regions that are involved in limbic seizures (hippocampus and amygdala) and midline thalamic sites with known connectivity to the amygdala and hippocampus (Herkenham, 1978; Krettek & Price, 1978; van Groen & Wyss, 1990; Turner & Herkenham, 1991; Ray et al., 1992; Su & Bentigvolio, 1990; Dolleman-van der Weel & Witter, 1996; van der Werf et al., 2002; McKenna & Vertes, 2004) to study the potential role that these sites play in seizure initiation and spread. The thought that the MD region may play a central role in limbic seizure activity is based on the key observations that it is always from the beginning involved in electrographic seizure activity involving the hippocampus (Bertram et al., 2001), and, as demonstrated in this study, that pharmacologic manipulation of this area has a clear effect on electrographic limbic seizure activity. The hypothesis is further supported by the observation that direct seizure induction from this site results in almost immediate spread of seizure activity as evidenced by the rapid motor involvement. Because MD has such a high afterdischarge threshold, however, it is unlikely that this region of the thalamus acts as a seizure initiator. However, once recruited into a seizure, it could, through its connections recruit other regions. This possibility requires further study.
As noted earlier, other investigators have examined the potential role of the MD nucleus in seizures. In those experiments they evaluated the effect of drug infusion into the MD region on the behavioral expression of induced seizures that was similar to ours (Patel et al., 1988; Hirayasu & Wada, 1992; Cassidy & Gale, 1998). These studies demonstrated a consistent effect on seizure behavior. Unfortunately, EEG was not recorded to determine the effect on seizure physiology, so one was not able to determine if the altered seizure behavior was the result of a treatment effect on electrographic seizure activity or of changes in seizure spread. The present study suggests the MD region can influence both. However, other studies examining the role of MD and its thalamic neighbors in other types of acute seizures (systemic pentylenetetrazole) found that infusion of GABAergic agents into the region significantly worsened the seizures (Miller et al., 1989; Miller & Ferrendelli, 1990). These observations emphasize that the role of any region in a seizure will depend on the seizure and its associated circuitry, as pentylenetetrazole-induced seizures are considered more of a model for generalized seizures.
There have also been studies in the past examining the effect of lesioning the thalamus on seizures induced by electrical stimulation of the amygdala. These reports, all from the same laboratory, showed a mixed effect on the kindling process as well as on established kindled seizures (McCaughran et al. 1978; Hiyoshi & Wada, 1988a, 1988b). Review of the studies, specifically the placement of the electrolytic lesions, suggested that the lesions were never complete and occasionally only marginally involved the MD nuclei. The data from the present study did show that the thalamic effects can be quite focal, which may explain the relatively mild effects of incomplete lesions. One study of kindling in the massa intermedia of the thalamus (a much broader midline region in the thalamus than examined in the present study) had similar results to ours. They did not examine the specificity of the midline region to show that the effect was strictly localized to a small region in the thalamus (Mori & Wada, 1992).
A similar functional anatomy for absence seizures, with the cortex providing the drive and the thalalmus supplying a necessary organization was proposed and strongly supported by studies using a feline model of absence seizures that was carried out several decades ago (Avoli & Gloor, 1982). They induced seizures by the focal or systemic application of penicillin, and, as in the present study, they found linked activity between the cortex and thalamus (in this case the lateral relay nuclei). They also found that both regions were necessary for the development of well-organized spike and wave activity. Of note, they could only drive the seizures from the cortex by penicillin application. In other words, the cortex provided the excitatory drive and the thalamus organized the activity. More recently, Meeren et al. (2002) demonstrated in a rat model of spontaneous spike and wave seizures a similar interaction between the cortex and thalamus, with the cortex likely providing much of the drive to initiate the seizures. The present results for limbic seizures are very similar. One is tempted to take these observations from two distinct seizure types and generalize them into a prototypical seizure model that is built around thalamocortical circuits. Each component in such a hypothesized circuit plays a distinct role in the seizure, and the physiology of the overall seizure activity is dictated by the physiology of the individual components (e.g., spike and wave activity or tonic activity).
Such a division of function and physiology in a seizure circuit may also suggest that there are potentially separate targets for therapy with entirely different physiology and pharmacology. Thus it is important to know what the circuits are, and more importantly, where control points lie so that therapy can be directed at those points and not globally as it is done now with systemically administered drugs. In this study, and in others, the MD and perhaps some of its midline thalamic neighbors appear to function as a control point. Pharmacological manipulation of this area modulates the duration of the seizure (primary circuit) and the behavioral severity of the seizure (seizure spread).
Although one can speculate about the mechanisms underlying the effect of intervention on seizure duration, the mechanisms of seizure spread are still unclear. The early involvement of the MD nucleus in limbic seizures and the rapid generalization of the seizure following stimulation of this midline region suggest that it is significantly involved, but there are a number of other hypotheses. One hypothesis would focus on the MD/midline nuclei as a primary path of spread. In considering its known connections (projection and reciprocal) it is easy to construct a scenario in which seizure activity would spread through the MD nucleus to the neocortex (Krettek & Price, 1977; Groenewegen, 1988; Ray & Price, 1992; Kuroda et al., 2004). The MD-limbic connections get the seizure started, and the seizure then spreads to other sites to which this thalamic nucleus is connected. Against the MD as a primary route of spread is the high afterdischarge threshold, an observation that raises the possibility that the drive for the seizure will more likely come from elsewhere. Another current hypothesis routes the seizure spread through the parahippocampal gyrus to other regions of the brain (Kelly & McIntyre, 1996; Kelly et al., 2002; Zhang et al., 2001). This scenario is based on the connectivity of this region, as well as on experimental evidence suggesting the spread of seizures through this site following stimulation. The amygdala similarly has widespread connections and could also serve as a path for generalization (Krettek & Price, 1978; Amaral & Price, 1984; Kita & Kitai, 1990). This issue needs further investigation.
There are several recent studies that, at first glance, are less supportive of the present conclusions. In a study examining the link between temporal lobe seizures and the thalamus in humans Guye et al. (2006) showed that there was thalamic involvement, but that the temporal relationship was much more variable. Although there were a few seizures that appeared to have the strong linkage between limbic sites (hippocampus and entorhinal cortex) and the thalamus, in other seizures there was less apparent synchrony at the time of seizure onset. However, the recordings came from multiple sites in the thalamus, including the pulvinar and several of the lateral relay nuclei with only a small number from the MD (three total in the MD and ten in the pulvinar and lateral nuclei). There were a few seizures that had a very tight linkage, but it was never made clear in the displayed EEGs where in the thalamus any given recording originated so that no conclusions can be drawn regarding the degree of linkage between a limbic site and a particular thalamic nucleus from the data as presented. It was clear that there was very good synchronization in at least one seizure, but specific details about the recording sites were not provided. As we have shown in this study, the relationship between the thalamus and the limbic sites varies with the thalamic nuclei, and if the recording site is in the "wrong" thalamic nucleus, the activity may be very poorly synchronized. Blumenfeld et al. (2007) have examined the relationship between the amygdala and the MD during the kindling acquisition. They showed a later involvement of the thalamus than what we have seen, an observation that would call into question our observations or suggest that our results may be more specific to the hippocampus. Reviewing their techniques, they used animals that were much smaller than ours, and the stereotactic coordinates used would put the thalamic electrodes on the outside edge of the MD. Assuming, as we have seen ourselves that placement can vary by as much as a half millimeter and that the target in the younger animals was smaller, it is possible that many of the electrodes were lateral to the MD. As we have seen thalamic involvement is highly focal, so that slight shifts can make large differences in recordings. Although they reported confirmation, electrode location was not shown.
In conclusion, this study has demonstrated that the MD/dorsal midline thalamic region is an integral part of the limbic seizure circuit and may play a role in seizure spread. Synaptic modulation in this region can alter limbic seizure activity, an observation that may have implications for targeting therapy. The kindling studies suggest that it is likely that the primary drive to initiate limbic seizures comes from the limbic sites, with the MD region acting as a key control point. Finally the data strongly support the hypothesis that, in the initial stages of seizure activity, thalamic involvement is specific to the MD region.