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Purpose: Mesial temporal lobe epilepsy (MTLE) is a common form of epilepsy that affects the limbic system and is associated with decreases in memory and cognitive performance. The medial prefrontal cortex (PC) in rats, which has a role in memory, is associated with and linked anatomically to the limbic system, but it is unknown if and how MTLE affects the PC.
Methods: We evoked responses in vivo in the PC by electrical stimulation of the mediodorsal (MD) and reuniens (RE) nuclei of the thalamus at several time points following status epilepticus, before and after onset of spontaneous seizures. Kindled animals were used as additional controls for the effect of seizures that were independent of epilepsy.
Results: Epileptic animals had decreased response amplitudes and significantly reduced recruiting compared to controls, whereas kindled animals showed an increase in both measures. These changes were not associated with neuronal loss in the PC, although there was significant loss in both the MD and RE in the epileptic animals.
Conclusions: There is a significant reduction in the thalamically induced evoked responses in the PCs of epileptic animals. This finding suggests that physiologic dysfunction in MTLE extends beyond primary limbic circuits into areas without overt neuronal injury.
The prefrontal cortex (PC) is associated with memory functions, as well as higher-level functions such as executive processing and decision-making (Deacon et al., 2003; Macrae et al., 2004; Price, 2005). The PC is reciprocally connected to limbic structures known to be involved in and affected by MTLE (Margerison & Corsellis, 1966; Vertes, 2004; Hoover & Vertes, 2007). Despite these links, the degree to which the PC is affected in MTLE is unknown. Positron emission tomography (PET) studies have shown signs of glucose hypometabolism in the PCs of MTLE patients (Jokeit et al., 1997; Takaya et al., 2006). However, the basis for these changes is unclear.
In this study, we sought to identify potential changes in the input to the medial PC from the midline thalamus in a rat model of MTLE, focusing on two distinct nuclei with overlapping prefrontal projections: the mediodorsal (MD) and reuniens (RE). These thalamic nuclei were chosen because they are closely connected to the limbic system (Giguere & Goldman-Rakic, 1988; Berendse & Groenwagen, 1991; Van der Werf et al., 2002; Gabbott et al., 2005), and are thought to participate in the initiation and spread of limbic seizures (Cassidy & Gale, 1998; Bertram et al., 2001, 2008).
To study the thalamic connection to the PC in epilepsy, we used trains of stimuli to induce the thalamic recruiting response, which is a progressive augmentation of the amplitude of the response wave that plateaus by the fourth stimulation in the train (Fig. 1). This response was first described in the early 1940s (Dempsey & Morison, 1942; Morison & Dempsey, 1943), and is strongly linked to midline thalamic stimulation and cortical excitation (Verzeano et al., 1953; Herkenham, 1986; Bazhenov et al., 1998). Because the PC shares connections with a variety of structures that are also networked to the midline thalamus, it was important to examine a cortical response that was strongly linked to thalamic input. The recruiting response is also a useful tool for examining the PC–midline thalamic connection because the maximal amplitude of the response in the recruiting rhythm is much larger than the response to single stimuli.
Figure 1. Recruiting responses. (A) Typical responses in the medial prefrontal cortex (PC) to midline thalamic stimulation in control animals. Responses on the left were elicited by mediodorsal (MD) stimulation (gray circle), whereas those on the right were elicited by reuniens (RE) stimulation (black circle). Arrows indicate depths at which responses were obtained. The recording electrode was moved from 1–4 mm below the cortical surface in order to identify areas of maximal response (see Table 1). Notice that the amplitude of the response increases until the fourth stimulation and then plateaus. (B) Responses recorded at stimulation intensities ranging from 15–70 V. Maximal responses did not increase significantly with higher stimulation intensities after 50 V. (Images of sections from Paxinos & Watson 1998.) (C) Demonstration of how parameters were measured. First and fourth response wave amplitudes were measured from the baseline previous to the stimulation, and then compared. Latencies were measured from the stimulation itself. Onset latency was measured to the start of the wave, and peak latency was measured to the highest point of the positive recruiting response.
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In this study, we found that there was a significant attenuation of thalamically evoked responses in the PC of epileptic rats. The attenuation evolved over a period of weeks during the post-SE period. Differences in seizure activity in the PC are not thought to be responsible for the change, as seizure activity was found in the PC in both kindled and epileptic animals. Neuronal loss in the PC was not found, although neuronal loss in the thalamus was confirmed. These findings have potential implications for prefrontal cortical function in MTLE.
The recruiting response is a thalamically induced cortical response that is specific to stimulation of the midline thalamic nuclei. Although the precise mechanisms of the recruiting response are largely unknown, the current theory is that the response is a result of the overlapping, or “nonspecific” connectivity of the midline thalamus (Morison & Dempsey, 1943; Verzeano et al., 1953; Schlag & Chaillet, 1962). The midline nuclei send monosynaptic projections to the entire medial PC, with each nucleus overlapping in their termination fields, forming a diffuse and interconnected network. These projections terminate in layers I, III, and VI of the PC (Barbas et al., 1991; Negyessy & Goldman-Rakic, 2005; Hoover & Vertes, 2007). Reciprocal connections return from the PC from layers II and V (Freedman & Cassell, 1991; Vertes, 2004). The theory is that the recruiting response is generated as this overlapping, reciprocal connectivity is able to incorporate and synchronize additional thalamic nuclei with each additional stimulation in a train. This is not to be confused with the similar “augmenting” response described in sensory and motor pathways, which is thought to be primarily cortically based (Herkenham, 1986; Castro-Alamancos & Connors, 1996). In addition, it is possible that other structures in the limbic system that share connections with both the PC and midline thalamus (such as the amygdala and hippocampus) may contribute to recruiting responses, but that possibility is, as yet, untested.
The changes we found in the PC responses are likely related to the thalamic neuronal loss, but the mechanism is unclear at this time. The altered responses changed over time, which parallels the many changes that occur following an acute neuronal injury. Cell death and recovery is an evolutionary process that occurs in several brain regions in our epilepsy model (Ben-Ari et al., 1981; Cavalheiro et al., 1982; Brandt et al., 2003; Dinocourt et al., 2003). The process involves the loss of neurons, loss of synaptic input, local reinnervation, and a spectrum of pre- and postsynaptic changes that alter individual and population excitability. The evolution of these changes have been studied extensively and in great detail in hippocampal and amygdalar circuits, among others, in epilepsy (Mangan & Bertram, 1998; Lehmann et al., 2000; Kumar & Buckmaster, 2006; Sloviter et al., 2006; Sun et al., 2007). PC neurons do not appear to die in our model of MTLE, but cell death in the midline nuclei likely causes a loss of first-line synaptic input, which, in turn, could lead to a level of denervation hypersensitivity (Koyama et al., 1993; Iizuka et al., 2002; Carlson et al., 2007; Viisanen & Perrovaara, 2007), and alterations in postsynaptic channel and receptor expression. During the subsequent weeks, reinnervation takes place and postsynaptic properties stabilize and return to an equilibrium state, causing the response to decrease. This evolution timeline would explain the rise in recruiting response that takes place after 7 days, and the subsequent decline. The exact mechanisms that cause these shifts in excitation are unknown.
The basis for the change following kindling is also unclear. As mentioned, the process of cell death that occurs in our epileptic model does not occur in our kindling model (Represa et al., 1993; Racine et al., 2002). Changesin kindled animals may include postsynaptic alterations as well as other mechanisms that allow for a better recruitment of the other thalamic nuclei that contribute to the recruiting response.
The finding that the peak latencies were not significantly different among groups in this study reflects some minor variance in the geometry of the peak from rat to rat, the slight variation in rise time to high and low amplitude peaks, and may also reflect the differences in postsynaptic properties to which we have alluded. The lack of any significant difference between onset latencies among groups suggests that latency is not a truly variable factor in observations.
The effect that the alteration in responses has on the normal function of the PC, if any, is also unclear. The PC is involved in various memory functions, including autobiographical, spatiotemporal, and working memory (Macrae et al., 2004), as well as emotional processing, executive functioning, and decision making (Barbas, 1995; Price, 2005). Lesions in the PC have given mixed but interesting results depending on the animal and the method used (Lacroix et al., 1998; Deacon et al., 2003), and abnormalities in the PC have been implicated as underlying factors in some psychiatric disorders, such as schizophrenia (Mitelman et al., 2005). Although the deficits in learning and memory that have been observed in patients with TLE (Glowinski, 1973; Viskontas et al., 2000; Voltzenlogel et al., 2006) have been rightly attributed to cellular damage in the hippocampus and other limbic structures, our results suggest that the effects of that damage may extend into the physiology, and perhaps functionality, of structures like the PC. This may even be the case in the kindled animals, in which some memory deficits have been suggested (Hanneson et al., 2001). More-detailed studies on this subject will be difficult, as it will not be easy to attribute a specific memory deficiency to the PC or any other limbic structure.
In conclusion, we have found an attenuation in PC responses to midline thalamic stimulation in epileptic rats, suggesting an impairment in their connection. This attenuation evolves following thalamic cell injury incurred by status epilepticus and may be attributable to cell loss. Other factors, including inputs to the PC from other structures and possible pre- and postsynaptic modifications may also contribute to the change in response. It is possible that this physiologic attenuation may be an indication of some form of functional deficiency in the PC, with implications for its involvement with learning and memory processes. These data reinforce our current understanding that a variety of higher-order functions require a complex circuitry, incorporating diverse groups of brain structures.