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Summary: Purpose: Functional imaging of animal models makes it possible to map the functional neuroanatomy contributing to the genesis of seizures. Pentylenetetrazol (PTZ)-induced seizure in rats, a relevant model of human absence and of generalized tonic–clonic epilepsy, was used to stimulate seizure activity within 30 s of administration while collecting continuous, high-resolution, multislice images at subsecond intervals.
Methods: Pilot studies were conducted to establish a quick and effective PTZ model for the imaging experiments. PTZ was then used to stimulate seizure activity in rats while collecting multislice functional MRI (fMRI) images from the entire forebrain at 4.7 Tesla. Ethosuximide (ESM) also was used to block seizure activity.
Results: Within 2–4 s of PTZ administration, a rapid increase in blood oxygen level–dependent (BOLD) signal intensity was noted in the thalamus, especially the anterior thalamic nuclei. Activity in the anterior thalamus peaked ∼15 s before seizure onset and was more than twofold greater than that in all other thalamic areas. The retrosplenial cortex showed a twofold greater increase in activity as compared with other cortical areas, also peaking at ∼15 s. The dentate gyrus was twice as active as other hippocampal areas but peaked just before seizure onset. Treatment with ESM blocked seizures, decreasing PTZ-induced activation in most forebrain areas. The anterior thalamus and retrosplenial cortex were essentially blocked by pretreatment with ESM.
Conclusions: The anterior thalamus, retrosplenial cortex, and dentate gyrus show the greatest increases in BOLD signal activity before seizure onset. Neurons in these areas may contribute to the neural network controlling the initiation of generalized tonic–clonic seizure.
Although data from chemical-stimulation, lesion, and electrophysiologic studies have identified multiple brain areas involved in the initiation and propagation of generalized tonic–clonic seizures (1), the precise neural pathway integrating these areas is unknown. Unlike focal seizures that originate from a single locus, generalized seizures arise from a complex integration of cortical and subcortical signals contributing to the characteristic synchronized paroxysmal brain activity (2). Coherence studies on EEG signals have focused on the reciprocal interactions between the cortex and thalamus in the genesis of generalized seizures. For example, Sherman et al. (3,4) identified the anterior thalamic nucleus as having a strong association with cortical EEG activity, acting as an essential gate in the propagation of pentylenetetrazol (PTZ)-induced generalized tonic–clonic seizure activity from subcortical areas to the cortex. Electrophysiologic studies like this are essential for understanding the genesis of generalized seizures because they provide a window of observation measured in tens to hundreds of milliseconds; however, they are limited to a small number of brain areas that can be sampled at a time. Functional magnetic resonance imaging (fMRI), although having a lower temporal resolution than electrophysiology, offers a method for observing global changes in neural activity that could be used to draw a map of putative neural substrates involved in the genesis of generalized seizures.
Recently the technical problems associated with imaging of fully conscious animals in ultra-high field MR spectrometers were resolved (5–7). Studying brain function in animals with the high temporal and spatial resolution of noninvasive MRI makes it possible to follow activation of neural pathways in a variety of behavioral and neurologic models ranging from sexual arousal in monkeys (8) to generalized absence seizures in rats (9,10). In the latter studies, activation of the corticothalamic circuitry responsible for generating and maintaining the spike–wave discharge in absence seizures was confirmed by using fMRI. The present study addresses the feasibility of using fMRI in fully conscious rats to follow the genesis of PTZ-induced generalized tonic–clonic seizures.
Van Camp et al. (11) performed the first simultaneous EEG and fMRI study on curarized rats given subcutaneous PTZ. Acquisition time for a multislice gradient-echo sequence with averaging was ∼1 min. Within 10 min of PTZ injection, animals showed behavioral signs typical of absence seizures, with facial twitching and head movement, followed 20–30 min later by episodes of tonic–clonic seizures. Bilateral spreading of both positive and negative blood oxygen level–dependent (BOLD) signal changes occurred over most of the cortex, hippocampus, amygdala, and brainstem. The areas of activation observed in these imaging studies were not unlike those identified in deoxyglucose metabolism studies and immunostaining for c-fos protein after PTZ seizures (12–17). Because of the slow onset and progressive development of seizure activity and the long image-acquisition times in this study, it was not possible to identify neural substrates involved in the genesis of PTZ-induced seizures. The present study was designed to image the functional neuroanatomy contributing to the genesis of generalized seizures by collecting multislice images at subsecond intervals. Because sensory feedback from the whole body contributes to the seizure threshold, these studies were done in nonparalyzed, fully conscious animals (18).
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The process of developing this model involved the validation and dose determination of the ICV injection, determining the stress and physiologic effects, and investigating the vehicle effects of the injection. From these dose–response studies, it was determined that the 1.5-mg dose of PTZ would predictably trigger clonic seizure activity ∼30 s after administration without significantly altering respiratory or cardiovascular function (Figs. 1 and 2, Table 1). A typical response consisted of a myoclonic jerk of the head and neck clonus starting in the face and spreading to forelimbs, culminating in whole body clonus and loss of posture with twisting–writhing. Tonus or tonic flexion with forelimb extension and hindlimb flexion was not observed in any of the animals tested at the 1.5-mg dose. EEG recordings showed repetitive hypersynchronous discharges which increased in frequency (Fig. 2). Behavioral and EEG measures of seizure activity lasted for ∼3–5 min, as confirmed by video monitoring (see supplemental material). Animals pretreated with 250 mg/kg of ESM 1 hour before PTZ injection showed complete blockage of behavioral and EEG measures of seizure activity (Fig. 2).
Figure 1. Pentylenetetrazol dose–response after intracerebroventricular injection. A: Onset of EEG spike discharges and behavioral clonic movements. B: Time to recovery.
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Figure 2. EEG recordings of pentylenetetrazol (PTZ)-induced seizures and blockade with ethosuximide (ESM). Shown are bilateral EEG traces at various intervals after PTZ injection in the absence and presence of ethosuximide. Samples taken at 30 s after an injection of PTZ show spike discharges that increase in frequency over time. Spike discharges were absent after ESM treatment.
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Table 1. Physiologic changes caused by vehicle, pentylenetetrazol, and pentylenetetrazol + ethosuximide
| ||Respiration Rate||Heart Rate||Blood Pressure|
|Vehicle (n = 4)|
| Pre Injection||141 (9.2) ||414 (28.2)||121 (7.2) |
| 30s Post Inj.||148 (11.7)`||398 (35.3)||117 (3.3) |
|PTZ (n = 4)|
| Pre Injection||135 (21.8)||409 (14.5)||119 (18.7)|
| 30s Post Inj.|| 177 (31.1)*||418 (33.2)||106 (23.9)|
|ESM+PTZ (n = 4)|
| Pre Injection||124 (17.3)||409 (17.0)||113 (32.7)|
| 305 Post Inj.||138 (30.3)||413 (22.9)||108 (28.5)|
To control for the effect of restraint stress and auditory stimulation on seizure threshold, four animals were surgically prepared as described and secured in a commercially available MR-compatible restraint device with built-in imaging coils (Insight Neuroimaging Systems, Worcester, MA, U.S.A.). In addition, a catheter was placed in the femoral artery, a pneumatic thoracic force transducer positioned over the chest, and a pulse oximeter (Nonin Medical, Plymouth, MN, U.S.A.) connected to the tail. While restrained and exposed to a taped recording of the noise from an imaging experiment, animals were injected ICV with 1.5 mg of PTZ in 20 μl of artificial CSF. Heart rate, blood pressure, respiratory rate, and EEG were continuously recorded. Again, seizure activity was routinely elicited within ∼30 s from the time of PTZ injection. No significant change in blood pressure or heart rate within 30 s of the ICV injection was seen in vehicle controls, PTZ-treated animals and PTZ-plus-ESM animals. The respiration rate increased in the first 30 s from 135 ± 13 to 177 ± 23 (p < 0.01) for the PTZ-treated group (Table 1) but remained unchanged for control and ESM-treated animals.
Imaging of ICV vehicle injections of 20 μl of artificial CSF produced changes in signal intensity along the cerebroventricles immediately after injection. Analysis of this signal change showed that the injection volume spread down the ventricular system in <3 s from its site of origin in the lateral ventricle. This vehicle injection had a modest but significant 2–3% increase in BOLD signal in all of the regions of interest (ROIs). The increase in BOLD signal was transient, lasting for only 5–7 s. Ipsilateral and contralateral analysis of bilateral ROIs showed no lateralization effect from the unilateral injection, including the anterior cingulate nearest the cannula. Although the cannula was placed in the rostral end of the lateral ventricle, the first ROIs to activate with PTZ alone were more caudal: the anterior thalamus and retrosplenial cortex (Fig. 4). Data from the vehicle injection indicate that vehicle has a minimal contribution to the increases seen after PTZ administration.
Figure 4. Pentylenetetrazol (PTZ)-induced changes in blood oxygen level–dependent (BOLD) signal intensity over time. Shown are composite time course data for PTZ-induced changes in BOLD signal in the absence (n = 5) and presence (n = 5) of ethosuximide. Changes are presented as an aggregate of whole-brain activity (top panels) and with subsets of major brain areas (lower panels).
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An activational map showing PTZ-induced positive BOLD signal changes is shown in Fig. 3. All major brain areas (e.g., thalamus, hippocampus, amygdala, and cerebrum) showed a significant increase in BOLD signal (Fig. 4). The most salient feature in the forebrain activation pattern was the rapid and robust increase in BOLD signal in the anterior thalamus, retrosplenial cortex, and dentate gyrus (Fig. 4). Within 3–4 s of PTZ injection, the anterior thalamus showed a rapid rise in BOLD signal, peaking at ∼15 s before seizure onset. Within 4–6 s of PTZ injection, the retrosplenial cortex was activated, also peaking at ∼15 s before seizure (Fig. 4). The anterior thalamus and retrosplenial cortex stood out because their change in BOLD signal intensity was more than twofold greater than that in other thalamic and cortical areas. Activity in the dentate gyrus was also twice as high as in other hippocampal areas but peaked ∼25 sec just before seizure onset (Fig. 4). The amygdala in general was activated by PTZ treatment, but no particular nuclear area distinguished itself.
At the onset of generalized seizure activity, a decrease in BOLD signal intensity was found for most ROIs (Fig. 4). In the case of the parietal cortex and retrosplenial cortex, this decrease was precipitate and robust. Indeed, all cortical areas showed robust decreases in BOLD signal during seizures that exceeded most other forebrain areas. The thalamus, particularly the anterior and dorsolateral areas, appeared to sustain their BOLD activation during seizures. The same was true of the dentate gyrus.
The ESM blockade of PTZ-induced seizures occurred in the face of modest increases in BOLD activity in all of the major brain areas (Fig. 5). The onset of activity in all ROIs was delayed by 10–12 s as compared with PTZ treatment alone. The medial dorsal thalamus was still responsive to PTZ in the presence of ESM, whereas the anterior and lateral dorsal thalami were effectively blocked (Fig. 4). All cortical areas showed sustained activity over the first minute of the data-acquisition period after PTZ treatment. Both the dentate and CA1 showed sustained activity, but CA3 collapsed into negative BOLD signal ∼40 s after PTZ treatment. Interestingly, only the cortical nucleus of the amygdala showed an immediate response to PTZ in the face of ESM blockade that was similar to PTZ alone.
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This study describes a method for predictably triggering generalized clonic seizure activity in awake rodents that is compatible with BOLD imaging. The acquisition of continuous, high-resolution, multislice images at subsecond intervals makes it possible to image neural substrates activated by PTZ before the onset of seizure activity. These PTZ-sensitive brain areas are all potential candidates contributing to the genesis of generalized seizures. Based on their rapid and robust change in BOLD signal intensity, the hippocampus, particularly the dentate gyrus, anterior thalamus, and the retrosplenial cortex, appears to play a prominent role in triggering seizure activity. Pretreatment with ESM delays the onset of activity in these areas and reduces their BOLD signal change. The anterior thalamic nuclei are essentially blocked as their activity is decreased from a 20% change in BOLD signal intensity to <2%.
The sensitivity of hippocampal and cortical neurons to PTZ-induced seizures was shown in the landmark article of Morgan et al. (13) measuring expression of c-fos messenger RNA and c-Fos protein. Several subsequent articles using immediate-early gene expression as a measure of neuronal activity underscored the sensitivity of these brain areas to PTZ-induced seizure (14,29). Adult rats limited to clonic seizures, as reported here, show strong c-Fos labeling in the different hippocampal subfields, piriform, cingulate, entorhinal, olfactory and parietal cortices, septum, amygdala, dorsal striatum, globus pallidus, and hypothalamus (15,17). This pattern of neuronal activation is much greater than that reported here by using BOLD imaging and most likely reflects the sampling time. The BOLD activation map show in Fig. 4 reflects neuronal activity before the onset of seizure, whereas measures of c-fos activity are routinely acquired 1–4 h after seizure. The low-to-moderate BOLD-activation pattern in areas like the piriform, entorhinal, and olfactory cortices would suggest they are not involved in the genesis of seizures but are more involved in the expression and maintenance of seizures.
The robust activation of the anterior thalamic area is not unexpected because it is know to be a critical neuroanatomic substrate, together with the mammillary bodies, in the regulation of PTZ-induced seizures. Multiunit electrical activity increases dramatically in the mesencephalon and thalamus ∼8–10 s before the onset of paroxysmal EEG discharges in cats treated with intravenous PTZ (30). Conversely, mesencephalic and prethalamic lesions reduce PTZ-induced electrical activity in cats (31), suggesting an ascending mesencephalic/thalamic pathway contributing to the genesis of generalized seizure. Studies by Mirski et al. (32–34) extended this early work in cats to focus on an epileptogenic pathway in guinea pig consisting of the mammillary bodies and anterior thalamus, as critical for PTZ-induced seizure activity. Manipulating the mammillary bodies and their connections to the anterior thalamus via the mammillothalamic tracts, with lesions, chemical injections, and frequency-specific electrical stimulations, blocks or reduces PTZ seizures. Similar experimental manipulations of the anterior thalamus also block or reduce PTZ seizures (35). The anterior thalamus appears to be the gateway to the cortex regulating PTZ-induced paroxysmal electrical activity, as evidenced by EEG coherence studies (3,4).
Given the findings in cats and guinea pigs reporting a mesencephalic/limbic pathway contributing to the genesis of PTZ-induced seizures, it was surprising to observe that the mammillary bodies (not shown; Figs. 3 and 5 activation maps) in this study were ostensibly devoid of BOLD signal activity. Several possible explanations account for this discrepancy. In the present study, PTZ was given in the lateral cerebroventricle in fully conscious rats, triggering seizures in seconds. In other studies, PTZ was always given intravenously in cats or intraperitoneally in guinea pigs under different experimental conditions with variable delays in seizure onset. The differences in drug administration, animal species, and experimental preparations may all contribute to this discrepancy.
The possibility exists that the mammillary bodies are not as critical to seizure initiation as once thought. In this imaging study, it can be observed that areas immediately adjacent to the mammillothalamic tracts (e.g., posterior hypothalamus, midline thalamic nuclei, ventromedial and anteromedial thalamus) are activated by PTZ before seizure onset. If these areas are important in the initiation of seizures, any electrolytic lesion aimed at the mammillothalamic tracts might affect their function and reduce seizure activity. This possibility was noted by Mirski and Ferrendelli (36). In an earlier publication, these authors reported no apparent increase in neuronal metabolic activity in the mammillary bodies as measured with [14C]2-deoxyglucose uptake in guinea pigs despite PTZ-induced EEG epileptiform discharges (37). However, when pretreated with ESM followed by PTZ, a significant accumulation of label occurred in the mammillary bodies, suggesting that this brain area may be involved in the anticonvulsant action of ESM. The findings from this metabolic activation study were corroborated here, as PTZ alone caused no change in BOLD signal intensity in the mammillary bodies, but in the presence of ESM, a significant increase in signal contrast was noted.
No evidence from our studies indicates that cortical activation precedes subcortical activation during the genesis of PTZ-induced generalized seizures. Data from sleep–wake studies and seizure studies point to a corticothalamic connection regulating the generation of cortical EEG patterns (38,39). Partial coherence estimations show that the cortex and anterior thalamus are synaptically correlated during cortical EEG and behavioral seizure expression (4). Time-delay calculation using EEG activity suggests the anterior thalamic nucleus leads in propagation of epileptiform activity (3).
The decrease in BOLD activity during the start of the clonic episode (∼30 s; Figs. 4 and 5), particularly in the cortical areas, is noteworthy and most likely does not reflect a decrease in neuronal activity. Caspers and Speckman (40) collected simultaneous recordings of paroxysmal electrical activity and tissue po2 at the surface of the cerebral cortex during PTZ-induced seizure. Seizure initiation was always preceded by a decrease in tissue po2. This transient tissue hypoxia caused by PTZ seizure can also be measured by a change in reduction/oxidation state of cytochrome oxidase in rat brain before systemic changes in blood pressure or heart rate (22). The presence of the resulting paramagnetic deoxygenated hemoglobin would cause proton dephasing and a decrease in BOLD signal intensity (41). The most dramatic negative BOLD signal at the putative onset of seizures occurred in the parietal and retrosplenial cortices and the basolateral amygdala. This would suggest that these areas have an exceedingly high PTZ-induced metabolic demand without appropriate compensatory blood flow.
The general effect of ESM blockade of PTZ-induced seizure is an overall reduction in BOLD signal intensity. The cortices and hippocampal subfields all show significant and sustained activation above baseline but without initiation of seizures. As noted earlier, a key neural substrate in PTZ-induced generalized clonic seizures is the anterior thalamus. The suppression of PTZ-induced changes in BOLD activity by ESM in this brain area may be responsible, in part, for the blockade of seizures. Limbic and association cortices connect to the dentate gyrus of the hippocampus through the angular bundle of the entorhinal cortex. In turn, the hippocampus has direct efferent connections to the anterior thalamus. Reducing the level of activity in the neural populations connecting to the anterior thalamus may be sufficient to suppress seizure activity. It also is possible that ESM blockade of the anterior thalamus prevents feed-forward recruitment and synchronization of limbic and association cortices critical for seizure initiation.
In summary, this study shows it is technically feasible to use fMRI and BOLD imaging to follow changes in brain activity associated with the genesis of generalized clonic seizures. The fact that these studies can be done in awake animals is a major advantage because it avoids the confound of anesthesia. From these studies, it was possible to generate activational maps of the forebrain identifying neural substrates that may be directly involved in the genesis of PTZ-induced generalized clonic seizures. The anterior thalamic nuclei and the dentate gyrus of the hippocampus were distinguished from other brain sites by their rapid and robust activation profile. The prominence of these two areas in the neuroanatomy of generalized seizures is well documented. One potentially important finding gleaned from these imaging studies is the observation that the retrosplenial cortex is more active than other cortical areas before seizure onset. Its rapid and robust activation profile and dramatic collapse in BOLD signal intensity coinciding with the start of seizure suggest a brain area of high metabolic activity. The anterior thalamus, particularly the anteroventral nucleus and its caudal extension, the lateral dorsal nucleus, have extensive reciprocal connections with the retrosplenial (posterior cingulate) cortex (42). Of all of the midline cortical areas, the retrosplenial has the greatest efferent association network extending rostrally to the anterior cingulate and prelimbic areas and caudally to the presubiculum. Hence the retrosplenial cortex may play a critical role in integrating thalamocortical activity during the initiation of generalized clonic seizures.