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- Materials and Methods
Purpose: Neural reorganization and interictal behavioral anomalies have been documented in people with epilepsy and in animal seizure models. Alterations in behavior could be due to somatosensory dysfunction. This study was designed to determine whether seizures can lead to changes in somatosensory representations and whether those changes are persistent.
Methods: Twice-daily seizures were elicited by delivering 1 s of electrical stimulation through carbon fiber electrodes implanted in both the corpus callosum and sensorimotor neocortex of young adult male Long-Evans rats until a total of 20 seizures were elicited. Either 1–3 days or 3–5 weeks following the last seizure, functional magnetic resonance imaging (MRI) was used to image the brain during electrical stimulation of each forepaw independently.
Key Findings: Forepaw stimulation in control rats resulted in a focused and contralateral fMRI signal in the somatosensory neocortex. Rats that had repeated seizures had a 151% increase in the number of voxels activated in the contralateral hemisphere 1–3 days after the last seizure and a 166% increase at 3–5 weeks after the last seizure. The number of voxels activated in response to forepaw stimulation was positively correlated with the duration of the longest seizure experienced by each rat. The intensity of the activated voxels was not significantly increased at either time interval from the last seizure.
Significance: The increased area of activation in somatosensory cortex, which is persistent at 3–5 weeks, is consistent with previous observations of larger motor maps following seizures. Seizure-induced changes in the functioning of sensory cortex may also contribute to interictal behavioral anomalies.
The mammalian neocortex is organized into topographic maps (Kaas, 1997). People with epilepsy have been shown to have differently organized sensory (Uematsu et al., 1992; Urasaki et al., 1994) and motor (Uematsu et al., 1992; Urasaki et al., 1994; Lado et al., 2002; Branco et al., 2003; Chlebus et al., 2004; Labyt et al., 2007) maps. These changes in the topographic maps may be associated with interictal behavioral deficits in planning and executing voluntary movements that some people with epilepsy display when the seizure focus is in the frontal lobes (Helmstaedter et al., 1996; Matsuoka et al., 2000; Hernandez et al., 2002). Although data from people with epilepsy give insight into how seizures can change neocortical organization, many extraneous factors, including the use of anticonvulsant drugs or underlying pathology, can confound the interpretation. Moreover, seizure characteristics like seizure duration that may play a role in directing neocortical reorganization are often self-reported and may be unreliable (Cramer et al., 2002).
Experimental animal models of seizures have been useful in addressing how seizures affect the functional organization of various brain structures (Pitkänen et al., 2006). Seizures can induce changes in neocortical movement representations and behavior (for a review see Teskey et al., 2008). We have shown previously that seizure activity in the neocortex can result in larger motor maps of the forelimb (Teskey et al., 2002; van Rooyen et al., 2006; Ozen et al., 2008; Young et al., 2009) as well as nonforelimb areas (Henderson et al., 2011) and can lead to alterations in forelimb performance on specific motor tasks (Henry et al., 2008; Flynn et al., 2010). Because successful behavior relies on both motor and sensory information, altered sensory processing may contribute to the behavioral deficits seen following seizures. Regarding sensory processing, it has previously been shown in feline primary auditory neocortex that neuronal firing responses to various sounds are dramatically altered following experimentally induced repeated seizures in that structure (Valentine et al., 2004, 2005). Whether changes to the cortical processing of somatosensory information can be detected in response to experimentally elicited repeated seizures has not yet been reported.
One method of evaluating somatosensory processing is by acquiring functional activation maps using functional magnetic resonance imaging (fMRI) during somatosensory stimulation. fMRI has been useful in providing insight into neural plasticity following stroke, demyelination, and spontaneous seizures (Baron et al., 2004; Hodics & Cohen, 2005; Ward, 2005; Lidzba et al., 2006; Cousin et al., 2008; Pillai, 2010). fMRI detects local increases in MR signal intensity due to decreases in the concentration of deoxyhemoglobin [blood oxygen level dependent (BOLD) signal] (Logothetis et al., 2001), which is associated with increases in neural activity and increased blood flow (Tuor, 2009). Three main questions were addressed in this study. First, we examined if 20 repeated experimentally induced seizures of relatively short duration (non–status epilepticus) result in increased volume of neocortical somatosensory activation to forepaw stimulation and if the effect is persistent. We also examined if the duration of individual seizures is correlated with the volume of activation. We employed electrical stimulation of the forepaw to provide reliable and repeatable sensory input (Wang et al., 2006; Tuor et al., 2007) for producing activation maps measurable with fMRI. We hypothesized that the volume of activation during forepaw stimulation would increase in rats who had repeated seizures both 1–3 days and 3–5 weeks following the last seizure.
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Three novel findings are presented in this study. This study provides the first demonstration of both a short-term (days) and persistent (weeks) enhanced MR responsiveness to somatosensory stimulation following repeated experimentally induced seizures. In addition, this study is also the first to describe a strong positive relationship between seizure duration and the volume of the activation measured with fMRI. We showed that rats that had repeated seizures induced in the sensorimotor neocortex had a greater number of activated voxels in response to electrical forepaw stimulation. This increase in voxel number was present both in the 1–3 day group and in the 3–5 week group. A bilateral response to stimulation of one forelimb was also seen in some of the rats in the 1–3 day group. In addition, the seizure-induced changes in somatosensory activation to forepaw stimulation did not show degradation in volume at 3–5 weeks following the last seizure. This is similar to the findings by Ozen et al. (2008), where they showed that larger forelimb motor maps following repeated seizures persisted and did not degrade in size for the maximum time examined: 5 weeks. The persistence of an augmented activation response also supports the idea that these seizure-induced effects are not directly related to the seizure events themselves or are transiently expressed, but are related to the long-term changes caused by repeated seizures (Dennison et al., 1995; Racine et al., 1995). Taken together, these results indicate that in addition to larger forelimb motor maps (Teskey et al., 2002; van Rooyen et al., 2006; Ozen et al., 2008; Young et al., 2009) and nonforelimb motor maps (Henderson et al., 2011) as well as behavioral alterations (Henry et al., 2008; Flynn et al., 2010), repeated seizures also affect the organization of the somatosensory neocortex.
The close spatial and temporal relationships between neural activity and cerebral blood flow form the basis of fMRI as a measure of brain activity. Larger BOLD signals are associated with local decreases in deoxyhemoglobin, which are evoked by increased oxygen consumption. This is thought to reflect increased metabolic activity that is related to both local synaptic activity (Goense & Logothetis, 2008) as well as spiking activity (Lee et al., 2010; Young et al., 2011) and results in local increases in cerebral blood flow. Seizure-induced changes to electrophysiologic measures such as enhanced evoked potentials (Teskey et al., 2002) and increased auditory unit responses to sound (Valentine et al., 2004, 2005) suggest that the increased fMRI signal to somatosensory stimulation is due, at least in part, to changes in local neural responsiveness. There was no change in the intensity of the activated voxels following seizures, indicating that changes in responsiveness to forepaw stimulation reflect a recruitment of neighboring sensory areas rather than simply an increase in blood flow to previously responsive areas. However, it is possible that vascular changes still may have occurred to allow the increased number of voxels to be activated. Factors involved with vasodilation, including cyclooxygenase-2 (COX-2) and nitric acid synthase, have been shown to be upregulated following electroconvulsive seizures (Newton et al., 2003). Other vascular changes may have occurred as well, allowing for enhanced oxygen delivery and perhaps accounting for some of the difference in responsiveness of the somatosensory cortex following seizures. The efficiency of the vasculature may also be altered, potentially contributing to the greater volume of fMRI signal. The idea that angiogenesis might occur in response to a discrepancy between metabolic demand and oxygen availability is supported by the observation that capillary density increases following hypoxic conditions (LaManna et al., 2004), wheel running in rats (Kleim et al., 2002), and sensory and motor training (Black et al., 1991; Isaacs et al., 1992; Swain et al., 2003). Similarly, following electroconvulsive seizures, angiogenic factors including vascular endothelial growth factor, fibroblast growth factor, and neuropeptide Y are also upregulated (Newton et al., 2003). In addition, electroconvulsive seizures induced in the rat hippocampus have been shown to induce the formation of new capillaries (Hellsten et al., 2005; Pitkänen et al., 2006). In people with temporal lobe epilepsy, angiogenic factors such as vascular endothelial growth factor are overexpressed (Croll et al., 2004; Rigau et al., 2007). These previous observations support the idea that seizure-induced vascular changes may also occur, allowing for enhanced oxygen delivery to the somatosensory cortex during forepaw stimulation, thus partially accounting for our observation of increased fMRI responsiveness to forepaw stimulation. To summarize, it is likely that changes to both neuronal and vascular organization may account for our observation of an increased volume of activation to forepaw stimulation.
Seizure-induced synaptic potentiation may account for the strong positive relationship between seizure duration and the volume of activated sensorimotor cortex, given that affected neurons during a seizure fire in a highly repetitive and highly synchronous pattern. This synchronous firing likely incurs the Hebbian rule that neurons that fire in temporal and spatial proximity incur stronger synaptic connections (Teskey et al., 2002; Valentine et al., 2004). This may explain why seizure duration is positively correlated to the number of voxels activated during forepaw stimulation, as longer exposure of synchronized firing would more strongly wire affected neurons together. The induction of neocortical long-term potentiation (LTP) in Long-Evans rats and rats that have been genetically selected to be seizure-prone (FAST rats) have larger movement representation areas (Flynn et al., 2004; Monfils et al., 2004). As well, seizures have been shown to induce robust synaptic potentiation in the pathways involved in seizure development (Racine et al., 1972, 1995; Teskey et al., 2002). As the duration of each seizure increases, it is likely that there is more synaptic potentiation accounting for the positive correlation between the length of individual seizures and the volume of activation in response to forepaw stimulation.
Although seizure-induced synaptic potentiation may explain the size and occurrence of bilateral activation to forepaw stimulation following seizures, it is unclear why bilateral activation does not persist for up to 5 weeks, like contralateral activation. Regular use of the forelimbs and the absence of seizures driving bilateral forelimb clonus may account for why bilateral activation was not seen 3–5 weeks following seizures. Nudo et al. (1996) has shown in squirrel monkeys that had enlarged digit motor representations following skilled digit training was reduced after cessation of training. It is possible that increased volume of activation to forepaw stimulation may also decrease in the contralateral hemisphere due to continued use of the forelimbs. Bilateral activation is likely to be pruned out earlier than contralateral activation. Most receptive fields of the cortex of mammals respond to contralateral rather than bilateral or ipsilateral input (Mountcastle, 1957; Armstrong-James & George, 1988), and would likely not be activated as much during regular forelimb use.
In summary, we have demonstrated that repeated neocortical seizures result in a greater volume of activation to somatosensory stimulation that is persistent. We have also demonstrated a strong positive relationship between seizure duration and the volume of activation to somatosensory stimulation that occurs following seizures. These results are similar to the clinical observations that some people with epilepsy have differently organized sensory maps (Uematsu et al., 1992; Urasaki et al., 1994). Possible mechanisms underlying the observed increase in neurovascular responsiveness include synaptic potentiation and vascular changes. Future studies should aim to further elucidate the mechanisms responsible for seizure-induced increases in neocortical responsiveness in the sensorimotor cortex, as well as how these changes relate to alterations in interictal behavior.