Correspondence: Atsuo Fukuda, MD, PhD, Department of Neurophysiology, Hamamatsu University School of Medicine, 20-1 Handayama 1-chome, Higashi-ku, Hamamatsu, Shizuoka 431-3192, Japan. Email: firstname.lastname@example.org
In order to study how the formation of focal cortical malformations is attributed to perturbation of developmentally multimodal γ-aminobutyric acid (GABA) functions, we made a focal cortical freeze-lesion on a rodent cerebral cortex at P0 (postnatal day 0). The microgyrus was formed at P7. GABA neurons were accumulated in the region surrounding necrosis at P4. Cortical plate cells born at E17.5 gathered, surrounding the GABA neurons, forming the cell dense portions in layer 2 of the microgyrus. Ambient GABA level was increased in the area corresponding to populated GABA neurons at P4. A KCC2 expression was downregulated, whereas an NKCC1 expression was upregulated in both the gathering GABA and cortical plate neurons, suggesting these cells had high intracellular Cl− concentration rendering GABA action depolarizing. GABAA receptor activation was involved in Ca2+ oscillation in these gathering cells. In vivo blockade of GABAA receptor prevented the above characteristic pattern of cell accumulation and hence microgyrus formation. Thus, neonatal freeze-lesion causes characteristic accumulation of differential populations of neurons preceded by characteristic release of GABA at an early stage, which induces GABAA receptor-mediated depolarization and Ca2+ oscillation. This paracrine/autocrine GABA may underlie the formation of neocortical malformations such as polymicrogyria.
The major inhibitory neurotransmitter GABA necessarily evokes depolarization in the immature brain, in contrast to hyperpolarization in the adult brain. Because the GABAA receptor is a chloride-negative (Cl−) channel, this developmental switch of GABA actions from depolarization (Cl− efflux) to hyperpolarization (Cl− influx) is induced by changes in the transmembranous Cl− gradient, which is regulated by Cl− transporters (NKCC1 (sodium, potassium, chloride cotransporter), uptake; KCC2, extrusion). In addition, the release mechanism of GABA changes from non-vesicular and non-synaptic to vesicular and synaptic ones, so that GABA actions are generally tonic in immature neurons and are converted to phasic with development. As these tonic depolarizing (and excitatory on occasion) GABA actions are necessary for neurogenesis, differentiation, migration and synaptogenesis, we term the above-mentioned GABA actions as “developmentally multimodal GABA functions” (Fig. 1).[1, 2]
On the other hand, neocortical malformations such as polymicrogyria show the presence of cortical cells in heterotopic positions; this is often caused by disruption in neuronal migration.[3, 4] Therefore, perturbation of the mulitimodal GABA function during development may underlie etiology of cortical malformations. Indeed, GABAA receptor-mediated responses are anomalously depolarizing in human focal cortical dysplasia tissue.[5, 6] However, how mulitimodality of GABA functions is disturbed is yet to be elucidated and it requires experiments using animal models.
The focal freeze-lesion (FFL) in the cerebral cortex of newborn (P0) rats produces a microgyrus, a focal cortical malformation with a small sulcus and a three- or four-layered microgyric cortex, which resembles human four-layered polymicrogyria,[7-10] a clinical condition that results from abnormal neuronal migration. Here, by using FFL models of rodents, we have addressed abnormalities in developmentally multimodal GABA functions. A part of this original data has been published and is available on-line (http://cercor.oxfordjournals.org/content/early/2012/12/16/cercor.bhs375.full?keytype=ref&ijkey=i04VZWybp7NFEDX).
Abnormal Modal Shifts of Cl−Homeostasis Underlying Neocortical Malformation
Altered GABAergic functions that occur during early brain development are induced by changes in Cl− homeostasis and play important roles in neocortical development by modulating events, such as laminar organization and synaptogenesis (Fig. 1). A relationship between polymicrogyria pathogenesis and Cl− homeostasis ontogeny in the developing parietal cortex were observed following FFL on both rats and mice at P0. In the FFL area, NKCC1 mRNA expression levels are greater in the lesion center-surrounding areas at P4 compared with adjacent exofocal areas. In contrast, although KCC2 mRNA re-emerges in the surrounding structure, expression levels are weaker than in the exofocal cortex. After P7, KCC2 mRNA expression increases in the microgyric region to levels similar to the intact surrounding cortex. Subsequently, NKCC1 mRNA expression in the microgyric region decreases to levels similar to exofocal cortices at P14–21.
In addition to the rat model, we have established a FFL in glutamic acid decarboxylase-green fluorescent protein (GAD67-GFP) knock-in (KI) mice maternally injected with 5-bromo-2′-deoxyuridine (BrdU) to distinguish GABAergic and glutamatergic neurons, with identified birthdates, responsible for the formation of a FFL-induced microgyrus. This model indicated that the above expressional changes in Cl− transporters were exclusively induced in the microgyrus-forming GABAergic and E17.5-born glutamatergic neurons.
Distinct Accumulation of Gabaergic and Late-born Glutamatergic Neurons in the Developing Microgyrus
Because GABAergic and glutamatergic neurons are generated, migrate and settle differently in the cortical region at distinct stages, we next addressed how the microgyrus is formed by migration of these distinct types of neurons. In combination with classical birth date analysis to label late-born neurons using BrdU, we determined GABAergic neurons and E14.5- or E17.5-born cells during formation of the microgyrus at P4.
In the lesioned cortex, few E14.5-born BrdU-positive cells reached the area surrounding the necrotic center (not shown). In contrast, many E17.5-born BrdU-positive neurons accumulated in the area surrounding the necrotic center. Interestingly, GFP-positive GABAergic cells accumulated around the lesioned area including in the superficial area (Fig. 2). Quantitative analysis of the distribution of GFP-positive cells revealed that they also tended to occupy the inner rim within 50 μm of the FFL border rather than the outer rim 50–100 μm from the FFL border (P < 0.05). In contrast to GFP-positive cells, E17.5-born BrdU-positive cells were almost absent in the superficial region of the lesioned cortex (P < 0.001). In addition, in contrast to GFP-positive cells, E17.5-born BrdU-positive cells tended to occupy the outer rim rather than the inner rim (P < 0.05).
In the area surrounding the FFL, a Cux1-positive, cell-dense band was detected (Fig. 2), whereas Tbr1-positive cells were absent in the area surrounding the FFL (Fig. 2) at P4. In the FFL area, E17.5-born BrdU-positive cells accumulated around the necrotic tissue and most co-localized with Cux1 (Fig. 2, inset), but not with Tbr1 (Fig. 2). These results suggest that the majority of E17.5-born cells that accumulated in the area surrounding the FFL were Cux1-positive glutamatergic cortical plate cells.
Temporal Increases in Ambient GABA in the Developing Microgyrus
Previous studies have suggested that GABA released from GABAergic neurons has a paracrine action on immature neurons,[14, 15] which may affect radial[16-21] and tangential[22-24] migration during neural development. Therefore, we next examined the spatio-temporal changes in extracellular GABA levels in the FFL cortex at different stages by means of GABA imaging recently developed.
The extracellular GABA concentration in the FFL center was about 43 μmol/L, and in the area < 100 μm from the FFL border was about 26 μmol/L; both values were significantly higher than those in the more distant areas (>100 μm: 9–11 μmol/L; Fig. 3). We also examined glutamate, using the modified method in which glutamate is catabolized by α-ketoglutarate by L-glutamic dehydrogenase (GDH), resulting in the production of a fluorescent reduced form of β-nicotinamide adenine dinucleotide (NADH). As shown in Figure 3, at P4, the ambient glutamate levels in the areas nearer to the FFL (within lesion: 2 μmol/L; <100 μm: 9 μmol/L) were much lower than those in the areas further from the FFL border (>100 μm: 25–26 μmol/L; Fig. 3).
Since this characteristic GABA accumulation was not observed in P2 or P7 FFL cortex, we speculated that the temporally increased extracellular GABA released from accumulated GABAergic neurons in the lesioned cortex might play a role in abnormal neuronal migration during formation of the microgyrus.
Intracellular Ca2+ Transients Mediated by GABAA Receptor Activation in Microgyrus-forming Cells
Given the transient increase in ambient GABA concentration and the potentially elevated intracellular Cl− concentration due to reciprocal expressional changes in Cl− transporters, NKCC1 and KCC2, in the vicinity of the FFL at P4, GABAA receptor-mediated depolarizing action may be responsible for the spontaneous Ca2+ oscillation that is important for regulation of neuronal migration.[26, 27]
The frequency of spontaneous Ca2+ transients was significantly higher in the cells < 100 μm from the FFL border than in those at the further site (P < 0.01; Fig. 4). When the GABAA receptor antagonist bicuculline methiodide (BMI; 20 μmol/L) was bath-applied, the number of spontaneous Ca2+ transients was significantly reduced only within 100 μm of the FFL border (P < 0.01; Fig. 4). These results suggest that tonic GABAA receptor-mediated depolarizing activity produces Ca2+ transients in migrating cells that are forming the microgyrus.
Chronic in vivo Blockade of GABAA Receptors Perturbs the Microgyrus Formation
Next, we blocked GABAA receptors continuously in vivo after FFL to study whether the accumulation of GABAergic neurons, causing a focal ambient GABA gradient, underlies microgyrus formation. BMI was injected intraperitoneally from P2 to P4 after FFL. At P7, although a sulcus-like formation was observed on the surface of the brain, the microgyric appearance of three- or four-layered cortex was obscured (Fig. 5).
Quantitative analysis by cell counting revealed that the preferential accumulation of GFP-positive GABAergic cells in the inner rim versus the outer rim was abolished by intraperitoneal BMI injection (Fig. 5F, see also Fig. 2G). In addition, cell density in the superficial region and the inner rim was significantly decreased by BMI treatment, while that in the outer rim was increased (Fig. 5F). Likewise, the preferential accumulation of Cux1-positive cortical plate cells in the outer rim versus the inner rim was abolished by intraperitoneal BMI injection (Fig. 5G).
The temporally increased ambient GABA causes tonic GABAA receptor activation, possibly depolarization, of NKCC1-upregulated and KCC2-downregulated neurons, resulting in modulation of Ca2+ oscillations. These Ca2+ signals could differentially affect the migratory status of GABAergic (“go”) and E17.5-born Cux1-positive cortical plate cells (“stop”). Therefore, immature cortical plate neurons and GABAergic neurons anomalously migrate with distinct patterns to form the microgyrus. Thus, abnormally induced mulitimodal GABA functions could be involved in migration disorders that ultimately result in cortical malformations.
Original studies contributed to this review were supported by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science; and by Grants-in-Aid for Scientific Research on Priority Area and on Innovative Area from the Ministry of Education, Culture, Sports, Science and Technology, Japan. The authors thank all the contributors of original papers: C. Shimizu-Okabe, T. Kumada, T. Morishima, S. Iwata, T. Kaneko, Y. Yanagawa, S. Yoshida, A. Okabe, W. Kilb, K. Sato and H.J. Luhmann, and also thank T. Furukawa for excellent technical assistance.