In the current study, we performed whole-cell current clamp recordings from human cortical neurons in layer 2/3 of the human neocortex in order to characterize the diversity of L2/3 human neocortical neurons in epileptic foci with various etiologies in order to begin to elucidate the underlying mechanisms of hyperexcitability which are still mostly unknown. We differentiated neuronal subtypes based on their firing patterns and AHP kinetics or epilepsy subtype (malformation of cortical development (MCD) vs. other (non-MCD)). We found that L2/3 pyramidal neurons have diverse firing properties and action potential kinetics, with some neurons looking remarkably similar to LTS interneurons. We also saw that L2/3 pyramidal neurons could be split into those with fast AHPs and those without, medium AHPs (mAHPs). Based on these parameters, we were unable to significantly differentiate neurons based on firing properties indicating that AHP component kinetics alone do not dictate L2/3 pyramidal neuron firing in human epileptic cortical slices. We also report significant differences in intrinsic properties between MCD and non-MCD and control L2/3 pyramidal neurons and are the first to characterize that wash on of the proconvulsant drug, 4-aminopyridine (4-AP), leads to increased AP duration, less firing rate (FR) accommodation, and slowed down AHPs. Overall, the present study is the first to characterize the large variability of L2/3 human neocortical pyramidal neurons, to compare between L2/3 pyramidal neurons within the epileptic foci between MCD and non-MCD cases, to use control tissue from tumor patients without incidence of seizure, and to determine the influence of 4-AP on L2/3 pyramidal neuron intrinsic properties.
INTRODUCTION:Childhood epilepsy is a common and often devastating disease, and chronic seizures lead to a significant increase in premature mortality and developmental delay. Focal cortical dysplasia (FCD), a malformation of cortical development, is the primary cause of pharmacoresistant epilepsy in children undergoing resective surgery. There are no targeted medical or surgical therapies for FCD patients who have failed surgery or who are not surgical candidates.METHODS:Human tissue from patients with MCDs was resected during epilepsy surgery. Comparison tissue from CD1 mouse brains was also used. 400-µm brain slices were made with a vibratome. The slices were then incubated the slices in Fluo-8L, a low-affinity calcium-sensitive indicator for 40-180 minutes. Slices were imaged using an sCMOS imager at 15 fps on an upright microscope and standard patch-clamp rig with a custom-built temperature-controlled chamber with high aCSF flow rate (5-15mL per minute) to optimize tissue oxygenation. Network bursts are induced with a pro-ictal ACSF containing high potassium (8 mM) and 4-aminopyridine (100 micromolar).RESULTS:We developed a workflow for calcium imaging in human tissue as follows. Preliminary semi-manual analysis on each image stack was performed using ImageJ. Image stacks were motion corrected using the NoRMCorre algorithm and then processed using the calcium imaging analysis workflow (CaImAn) appropriate for 1-photon imaging. Hundreds of cells can be analyzed simultaneously. Ictal network activity may be induced with the pro-ictal ACSF, or with gabazine (1 mM, a GABA-A receptor antagonist, data not shown).CONCLUSION:We have demonstrated the feasibility of performing ex vivo calcium imaging to directly study the aberrant epileptic networks in human MCD tissue resected from children with intractable epilepsy. In future work, we will use this novel technique to uncover network anomalies underlying the epileptic networks in this disorder.
Drug-resistant epilepsy (DRE) is a prevalent problem in children that can lead to abnormal development and various psychiatric comorbidities. Malformations of cortical development (MCD) include focal cortical dysplasia, tuberous sclerosis complex and hemimegalencephaly, which are the most common pathologies among children who undergo surgical resection for treatment of DRE. These disorders share many histopathological features, including dyslamination of the cerebral cortex and enlarged neuronal somata. Recently, genetic mutations in the mammalian target of rapamycin (mTOR) signaling cascade have been shown to underpin most MCDs. Rodent models, including the RhebCA model, recapitulate histologic and physiologic aspects of human DRE. However, there have been few studies characterizing the developmental time point of the histological changes seen in MCDs. In this study, we use in utero electroporation to upregulate the Rheb protein (directly upstream of mTOR) in a focal area of the neocortex. We demonstrate that mTOR dysregulation leads to focal dyslamination and increased neuronal size that is histologically similar to MCD, which correlates to spontaneous recurrent seizures. We used immunohistochemistry to investigate neuronal lamination at several time points during development between E18 and P21 and show early differences in lamination that persisted through development. Furthermore, the increased axonal length associated with mTOR upregulation occurs early in development. Our study provides a time frame for the initial development of abnormal neuronal migration and cellular growth that occurs in MCDs, and our data supports that these anatomical changes may contribute to the formation of epileptic networks.
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