Genetic absence epilepsy: Effective connectivity from piriform cortex to mediodorsal thalamus
Introduction
Genetic absence epilepsy is a common form of childhood epilepsy [1] associated with absence seizures that are generalized epileptic events that cause a brief loss of consciousness [2]. Absence seizures are characterized by generalized spike–wave discharges (SWDs) (5–9 Hz in animal models, 3–5 Hz in humans) detected with electroencephalography (EEG) recordings. Animal studies have shown that the layer 5/6 of perioral somatosensory cortex likely generates absence seizure activity [3], [4], [5] that can be readily propagated via cortico-thalamic-cortico pathways [6], [7]. While there has been a strong focus on corticothalamic mechanisms of absence epilepsy, there has been less focus on the role of limbic structures in absence epilepsy. Recent studies have shown abnormalities in mesial temporal regions such as the hippocampus, amygdala, and piriform cortex in both animal models of absence epilepsy [8], [9], [10], [11], [12] and human patients with absence epilepsy [13], [14]. The piriform cortex is highly susceptible to hyperexcitable neuronal activity [15]. It also exhibits increased blood flow in Genetic Absence Epilepsy Rats from Strasbourg (GAERS), a well-established model of absence epilepsy [16], [17], [18], compared with nonepileptic controls (NECs) [8]. Increased blood flow was observed in the somatosensory cortex and ventrobasal thalamus as well as amygdala, entorhinal cortex, and hippocampus (CA2). This suggests that there may be a relationship between thalamocortical and limbic structures in absence epilepsy [12]. There is also clinical evidence of bilateral activation of the piriform cortex occurring synchronously with generalized SWDs in patients with absence epilepsy [13], however, it is unknown if there is any subtle changes in activity prior to the onset of seizures. Furthermore, the piriform cortex displays several anatomical pathways that link it to key regions associated with the absence epilepsy network [19] including unidirectional excitatory projections to the mediodorsal thalamus [20], [21], [22], [23], [24]. The mediodorsal thalamus is a higher-order thalamic nucleus that displays excitatory cortico-thalamocortical connections with the prefrontal cortex, supports it in cognition [25], and may play a potential role in absence epilepsy. The mediodorsal thalamus has demonstrated local pathological changes in the GAERS rat model [26] as well as increase in neuronal firing at the peak of SWDs in the Wistar Albino Glaxo from Rijswijk (WAG/Rij) rat model [27], another well-established animal model of absence epilepsy [28]. Therefore, the piriform cortex to mediodorsal thalamus pathway may contribute to or form part of the absence epilepsy network. In order to investigate whether this pathway is part of the absence epilepsy network, it is necessary to apply a brain connectivity measure.
Brain connectivity measures such as correlation and coherence describe whether two brain regions display similar neuronal firing or oscillatory activity with each other but do not inform whether one brain region is driving activity in the other [29], [30]. In this study, we use another form of connectivity named ‘effective connectivity’. Effective connectivity is defined as “…the influence one neural system exerts over another” [29] and can be applied to EEG and local field potential (LFP) data [31], [32]. Transfer entropy is a novel measure accounting for ‘effective connectivity’ and is considered to be equivalent to Granger causality, another effective connectivity measure [33], however, transfer entropy is capable of handling non-Gaussian distributed variables commonly seen in biological data. Transfer entropy has been used to quantify effective connectivity between neurons, and it has also been applied to LFPs to detect changes in connectivity between brain regions in different brain states [34], [35]. We used transfer entropy to establish a unidirectional estimate of effective connectivity to evaluate neural oscillatory communication between the piriform cortex to the mediodorsal thalamus. This pathway is a well-defined and validated pathway in the neuroanatomical literature, and we define this pathway as a strongly direct causal interaction. We implanted multichannel microelectrode arrays into the piriform cortex and mediodorsal thalamus to record LFPs in GAERS and NEC strains. Our frequency bands of interest were “wide” theta (2–12 Hz), beta (15–35 Hz), and gamma (36–80 Hz) as described in [36] and “narrow” theta (4–8 Hz) because of its reported early changes prior to absence seizure onset [37]. Across frequency bands, we hypothesize that the GAERS strain will exhibit increased effective connectivity, before and during generalized epileptogenic discharges, in the well-characterized neural pathway from piriform cortex to mediodorsal thalamus, compared with the NEC strain.
Section snippets
Experimental setup
Experiments were performed on adult male rats (aged 4–6 months) from the GAERS strain and a NEC strain that were anesthetized using systemic Intraperitoneal (I.P.) injections of urethane (20% vol/vol) until the rats were unconscious. Urethane is considered to be an unbiased anesthetic that affects both excitatory and inhibitory systems [38]. The rats were then placed in a stereotaxic frame with a thermoregulatory head pad to maintain body temperature at 37 °C. Burr holes were drilled over
Transfer entropy — theta effective connectivity
There was greater effective connectivity of pre-onset states versus the control states for “wide” theta band transfer entropy (t(4) = 2.4604, p < 0.05). There were no changes between pre-onset and control (t(2) = 1.921, p = 0.097) or onset and control (t(2) = 2.729, p = 0.056) for the piriform cortex and mediodorsal thalamus pathway. In the “narrow” theta band, there was greater effective connectivity in the pre-onset versus control states (t(2) = 3.313, p < 0.05) while there were trending
Discussion
In line with our hypothesis, we observed increased theta, beta, and gamma effective connectivity from the piriform cortex to the mediodorsal thalamus, during the onset of epileptic brain states. We also observed trending increase in theta effective connectivity during pre-onset brain states in this pathway. These findings suggest that this well-characterized neural pathway between piriform cortex and mediodorsal thalamus may be recruited as part of the ‘absence epilepsy network’.
Conclusions
The application of transfer entropy to determine the effective connectivity between subcortical structures that are directly connected in an epileptic animal model is a novel endeavor. With a global time lag estimate, it is simple to implement and can provide insight into how communication from one brain region to another occurs via functional spectral bands. From our preliminary findings, this method of investigating effective connectivity could be applied to the study of other epileptic
Author contributions
Authors had full access to all data and take responsibility of the presented data. Prof. Antonio G. Paolini and James C. Young designed and performed the experiments together. James Young analyzed all of the data acquired. All authors interpreted the data and were involved in drafting and revising of the article with respect to intellectual content. The study was supervised by Prof. Antonio Paolini and Prof. Graeme D Jackson.
Declaration of Competing Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationship that could be construed as a potential conflict of interest.
Acknowledgments
We thank Angela Lim for assistance in electrophysiology recording monitoring and aiding in transcardial perfusion and Dr. Helen Nasser for her assistance with performing histology. We also acknowledge Prof. Terence O'Brien and Dr. Pablo Casilla-Espinosa for providing GAERS and NEC rats for this study. The Florey Institute of Neuroscience and Mental Health acknowledges the strong support from the Victorian Government in particular the funding from the Operational Infrastructure Support Grant.
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