Are Ectopic Neurons a Red Herring in Localizing Seizure Foci?

Normotopic Cortex Is the Major Contributor to Epilepsy in Experimental Double Cortex.

Petit LF, Jalabert M, Buhler E, Malvache A, Peret A, Chauvin Y, Watrin F, Represa A, Manent J-B. Ann Neurol 2014;76:428–442.

OBJECTIVE: Subcortical band heterotopia (SBH) is a cortical malformation formed when neocortical neurons prematurely stop their migration in the white matter, forming a heterotopic band below the normotopic cortex, and is generally associated with intractable epilepsy. Although it is clear that the band heterotopia and the overlying cortex both contribute to creating an abnormal circuit prone to generate epileptic discharges, it is less understood which part of this circuitry is the most critical. Here, we sought to identify the origin of epileptiform activity in a targeted genetic model of SBH in rats. METHODS: Rats with SBH (Dcx-KD rats) were generated by knocking down the Dcx gene using shRNA vectors transfected into neocortical progenitors of rat embryos. Origin, spatial extent, and laminar profile of bicuculline-induced interictal-like activity on neocortical slices were analyzed by using extracellular recordings from 60-channel microelectrode arrays. Susceptibility to pentylenetetrazole-induced seizures was assessed by electrocorticography in headrestrained nonanesthetized rats. RESULTS: We show that the band heterotopia does not constitute a primary origin for interictal-like epileptiform activity in vitro and is dispensable for generating induced seizures in vivo. Furthermore, we report that most interictal-like discharges originating in the overlying cortex secondarily propagate to the band heterotopia. Importantly, we found that in vivo suppression of neuronal excitability in SBH does not alter the higher propensity of Dcx-KD rats to display seizures. INTERPRETATION: These results suggest a major role of the normo-topic cortex over the band heterotopia in generating interictal epileptiform activity and seizures in brains with SBH.

Commentary

Subcortical band heterotopia (SBH), also known as double cortex syndrome, is a cortical malformation associated with severe epilepsy.1 The characteristic feature of SBH is a band of gray matter within the subcortical white matter. This ectopic gray matter is made up of neurons that fail to migrate to their proper location in the cortical lamina during development. SBH results predominantly from mutations in the doublecortin (Dcx) gene, a microtubule associated protein expressed in proliferating and immature neurons of the cortex and hippocampus.2, 3 The majority of patients with SBH are female (X-linked pattern of inheritance); males with Dcx mutations present with lissencephaly. Patients with SBH often suffer from pharmacoresistant epilepsy and are poor candidates for surgical intervention. The interesting anatomical changes associated with SBH result from a specific interruption in cortical development.

During normal corticogenesis, immature neurons are generated in periventricular regions and migrate up into the cortical plate using multiple guidance cues from a variety of cell types to reach their proper location. In SBH, however, a subpopulation of neurons fails to migrate and remains stalled in the white matter tracts beneath the cortex.4 These neurons would normally integrate into the cortical circuitry and shape neuronal activation and network function. Without these neurons in place, the remaining cortical network may be significantly disrupted. Because patients with SBH are poor candidates for surgical intervention, intracranial or depth electrode recording is uncommon. Therefore, there are very little data to help identify the seizure focus in SBH. Are the ectopic neurons themselves or the normotopic cortex above the SBH the site of seizure initiation?

In this study, Petit and colleagues set out to identify the source of abnormal activity in SBH using a rodent model and high-density microelectrode array recording. SBH can be modeled in rodents using in utero RNA interference. Briefly, DNA constructs containing a short-hairpin RNA (shRNA) targeting the 3′ untranslated region of the Dcx messenger RNA (mRNA), or scrambled sequence controls, are injected into the lateral ventricles of embryonic day 15 Wistar rats. shRNAs binds to mRNAs of the target gene (Dcx) and lead to its degradation. DNA constructs encoding green or red fluorescent protein are also injected to allow identification of Dcx knock-down cells. A brief electrical voltage is discharged across the head of the embryos, allowing uptake of the DNA constructs into neurons in the subventricular zone and silencing of the target genes. This is a robust and reliable way to decrease the expression of Dcx protein during corticogenesis and has shown that formation of SBHs is due to loss of Dcx expression.

To identify the anatomical location of abnormal circuit function, the authors prepared acute cortical brain slices from postnatal day 13–17 Dcx-knockdown rats and used microelectrode array recording and elegant analysis to monitor electrical activity with high spatiotemporal specificity. Microelectrode arrays allow recording from multiple sites (in this case, 59) within a single brain slice. Each microelectrode, and its associated electrical activity, can be mapped onto a specific anatomical region. Interictal-like epileptiform (ILE) events were evoked via pharmacological blockade of GABA_A receptors, and their initiation and propagation were mapped using array recording. Somewhat surprisingly, ILEs were never initiated by the ectopic neurons but instead began in the normotopic cortex directly above the SBH. The ectopic neurons were integrated into the cortical circuit, however, as more than 90% of ILEs propagated from the normotopic cortex into the ectopic neurons in the SBH. These findings show that cortical hyperexcitability in Dcx-KD animals is a product of abnormal circuitry in the cortex above the SBH, rather than in the SBH itself.

To further identify the location of circuit dysfunction in this model of SBH, the authors examined in vivo hyperexcitability. Although Dcx-KD rats do not have spontaneous seizures, they are more sensitive to chemoconvulsant-induced seizures. To determine if this in vivo phenotype in Dcx-KD mice is due to neurons in the SBH or normotopic cortex directly above, the authors used a clever variation on their in utero electroporation approach. In a subset of experiments, DNA encoding a potassium channel was electroporated either slightly before, or simultaneously with, Dcx shRNA DNA. Expressing this potassium channel decreased neuronal excitability and therefore allowed the authors to address whether attenuating the excitability of neurons in the normotopic cortex (electroporation slightly before Dcx shRNA) or in the SBH (simultaneous electroporation with Dcx shRNA) affected a Dcx-KD rat's response to convulsants. In line with their in vitro results, decreasing the excitability of normotopic neurons, but not ectopic neurons, restored normal sensitivity to a convulsant.

Not only do these results shed light on a longstanding question in pathophysiology of SBHs, they also complement a number of recent studies examining similar questions in other diseases of cortical malformation. In both human and rodent models of cortical malformations, it is often the area surrounding a malformation that is the most hyperexcitable or likely to initiate a seizure.6–8 The cerebral cortex is defined by complex connectivity and balanced reciprocal synaptic transmission. Perhaps disrupting this balanced network is more detrimental than creating a new, unevolved, mutant structure? Furthermore, if ectopic neurons in malformations don't receive normal input, they may be electrically unresponsive. This study and others show that areas of malformation are integrated into active neuronal circuitry. This input, however, may be isolated from other normally coordinated inputs required to initiate ILEs and seizures. Also worth considering are the changes in activity-dependent maturation that ectopic neurons likely receive. Neurons outside of the developing cortical circuitry may not experience developmentally appropriate electrical and chemical signals.

The specific circuit level mechanisms that explain these interesting findings have yet to be determined but are essential to understanding the developmental alterations that lead to hyperexcitable cortical networks. This work also has relevance to neurosurgical approaches to treating cortical malformations. Perhaps lesions that were previously thought to be inoperable may not be the relevant site of ictal initiation. A blended strategy considering both the malformation and the normal home of those ectopic neurons may be more beneficial. Maybe the ictal focus you're looking for is hiding right next door…