Abstracts

Rationale: Focal cortical dysplasia (FCD) is one of the most frequent causes of refractory focal epilepsy, and are currently classified into three main types based on their morphological features upon histopathological examination. Their variability in morphology, location, and extension are major hurdles to early and accurate diagnosis and prognosis. Moreover, it is still unclear which mechanisms drive epileptogenicity in these lesions. Here, we used an animal model of cortical dysplasia (Benardete, Epilepsia 2002; 43, 970-982) to investigate the functional network properties and their response to a hyperexcitable challenge.

Methods: We studied the offspring of pregnant Wistar rats that were injected with either saline solution (Control n=21) or carmustine (BCNU n=26) (20 mg/kg i.p.) at 14 days of gestation. Ten animals per group were submitted to anatomical T2-weighted MRI at 30, 37, 44, and 51 postnatal days using a 7 T preclinical scanner (resolution = 67x67x80, 80 𝜇m³) to derive cortical thickness at the level of the primary motor cortex (M1) in a coronal slice. To confirm cortical cellular disorganization, we performed layer-specific immunofluorescence examinations of M1 in coronal sections of 3 control and 6 BCNU cryopreserved specimens at p30 (Foxp2 for layer V, Necab for layer IV, and NeuN for neurons). Finally, another group of Control (n=8) and BCNU (n=10) animals were used for in vitro calcium imaging to assess the activity and organization of intracortical circuits at p30. Brain slices were recorded using a stereoscope fluorescence microscope (Leica M205 FCA) coupled to a CCD camera. Each brain slice (230 𝜇m) was permeabilized with Fluo-4 AM at atmospheric conditions (CO2 5%, 32°C, O2 2%). Each slice’s activity was recorded according to the following sequence: basal with artificial cerebrospinal fluid (aCSF), pilocarpine (13 min total, subdivided as follows: 10 s basal-30 s pilocarpine 300 𝜇M, rest of aCSF perfusion), and KCl (13 min total, subdivided as follows: 10 s basal-30 s KCl 140 mM, rest of aCSF perfusion).

Results: Dysplastic cortexes at an early stage of development (p30) show macrostructural (Figure 1A, 1B) and microstructural differences characterized by evident delamination and neuronal dispersion (Figure 1C). Functionally, the dysplastic cortices showed a rearrangement of intracortical connectivity after the hyperexcitable stimulus, making them weaker and less connected, without returning to their basal state (Figure 2A). Furthermore, they show less stability in their internal network communication after the external hyperexcitable stimulus (Figure 2B).

Conclusions: Our results suggest that the disarranged structure of dysplasias may affect intracortical connectivity after an external hyperexcitable stimulus, which reduces their connections and renders them less dynamic.

Acknowledgments: We thank Nydia Hernández-Ríos, Ericka de Los Ríos and Juan Ortiz-Retana for technical assistance.

Funding: Please list any funding that was received in support of this abstract.: Funding provided by UNAM-DGAPA-PAPIIT (IN204720 for LC and IA200621 for HLM).