Junya Ichikawa

GABAergic excitation after febrile seizures induces ectopic granule cells and adult epilepsy.

Temporal lobe epilepsy (TLE) is accompanied by an abnormal location of granule cells in the dentate gyrus. Using a rat model of complex febrile seizures, which are thought to be a precipitating insult of TLE later in life, we report that aberrant migration of neonatal-generated granule cells results in granule cell ectopia that persists into adulthood. Febrile seizures induced an upregulation of GABAA receptors (GABAA-Rs) in neonatally generated granule cells, and hyperactivation of excitatory GABAA-Rs caused a reversal in the direction of granule cell migration. This abnormal migration was prevented by RNAi-mediated knockdown of the Na+K+2Cl− co-transporter (NKCC1), which regulates the excitatory action of GABA. NKCC1 inhibition with bumetanide after febrile seizures rescued the granule cell ectopia, susceptibility to limbic seizures and development of epilepsy. Thus, this work identifies a previously unknown pathogenic role of excitatory GABAA-R signaling and highlights NKCC1 as a potential therapeutic target for preventing granule cell ectopia and the development of epilepsy after febrile seizures.

Long-Range Axonal Calcium Sweep Induces Axon Retraction

Axon guidance molecules trigger a cascade of local signal in growth cones and evoke various morphologic responses, including axon attraction, repulsion, elongation, and retraction. However, little is known about whether subcellular compartments, other than axonal growth cones, control axon outgrowth. We found that in isolated dentate granule cells, local application of glutamate to the somatodendritic areas, but not the axon itself, induced rapid axon retraction, during which a calcium wave propagated from the somata to the axon terminals. The calcium wave and axon retraction were both inhibited by blockade of voltage-sensitive calcium channels and intracellular calcium dynamics. A combination of perisomatic application of calcium ionophore and depolarizing current injection induced axonal calcium sweep and axon retraction. Thus, perisomatic environments can modulate axon behavior through long-range intracellular communication.

The ratio of ‘deleted in colorectal cancer’ to ’uncoordinated-5A‘ netrin-1 receptors on the growth cone regulates mossy fibre directionality

Proper axonal targeting is fundamental to the establishment of functional neural circuits. The hippocampal mossy fibres normally project towards the CA3 region. In the hippocampi of patients with temporal lobe epilepsy and related animal models, however, mossy fibres project towards the molecular layer and produce the hyperexcitable recurrent networks. The cellular and molecular mechanisms underlying this aberrant axonal targeting, known as mossy fibre sprouting, remain unclear. Netrin-1 attracts or repels axons depending on the composition of its attraction-mediating receptor, deleted in colorectal cancer, and its repulsion-mediating receptor, uncoordinated-5, on the growth cone; but the roles of netrin-1-dependent guidance in pathological conditions are largely unknown. In this study, we examined the role of netrin-1 and its receptors in mossy fibre guidance and report that enhanced neuronal activity changes netrin-1-mediated cell targeting by the axons under hyperexcitable conditions. Netrin-1 antibody or Dcc ribonucleic acid interference attenuated mossy fibre growth towards CA3 in slice overlay assays. The axons were repelled from CA3 and ultimately innervated the molecular layer when hyperactivity was pharmacologically introduced. We first hypothesized that a reduction in netrin-1 expression in CA3 underlies the phenomenon, but found that its expression was increased. We then examined two possible activity-dependent changes in netrin-1 receptor expression: a reduction in the deleted in colorectal cancer receptor and induction of uncoordinated-5 receptor. Hyperactivity did not affect the surface expression of the deleted in colorectal cancer receptor on the growth cone, but it increased that of uncoordinated-5A, which was suppressed by blocking cyclic adenosine monophosphate signalling. In addition, Dcc knockdown did not affect hyperactivity-induced mossy fibre sprouting in the slice cultures, whereas Unc5a knockdown rescued the mistargeting. Thus, netrin-1 appears to attract mossy fibres via the deleted in colorectal cancer receptor, while it repels them via cyclic adenosine monophosphate-induced uncoordinated-5A under hyperexcitable conditions, resulting in mossy fibre sprouting.

Experimental febrile seizures induce age-dependent structural plasticity and improve memory in mice.

Population-based studies have demonstrated that children with a history of febrile seizure (FS) perform better than age-matched controls at hippocampus-dependent memory tasks. Here, we report that FSs induce two distinct structural reorganizations in the hippocampus and bidirectionally modify future learning abilities in an age-dependent manner. Compared with age-matched controls, adult mice that had experienced experimental FSs induced by hyperthermia (HT) on postnatal day 14 (P14-HT) performed better in a cognitive task that requires dentate granule cells (DGCs). The enhanced memory performance correlated with an FS-induced persistent increase in the density of large mossy fiber terminals (LMTs) of the DGCs. The memory enhancement was not observed in mice that had experienced HT-induced seizures at P11 which exhibited abnormally located DGCs in addition to the increased LMT density. The ectopic DGCs of the P11-HT mice were abolished by the diuretic bumetanide, and this pharmacological treatment unveiled the masked memory enhancement. Thus, this work provides a novel basis for age-dependent structural plasticity in which FSs influence future brain function.

Reelin promotes attachment of neuronal leading process to radial glia in dentate gyrus

KIAA0319 gene is associated with the onset of dyslexia. KIAA0319 encodes a transmembrane protein that is reported to be involved in the regulation of neuronal migration in the developing brain. Because KIAA0319 mRNA is also expressed in the adult brain, it may have other functions than the regulation of neuronal migration. In this study we examined the function of KIAA0319 on neuronal morphology. We found that primary cultured neurons expressing full-length KIAA0319 protein formed complex neurites and fine branches. On the other hand, expression of a mutant KIAA0319 protein that lacked the intracellular region had less effect. It was thus likely that KIAA0319 was involved in the regulation of neuronal morphology, presumably by interacting with a certain intracellular molecule(s). We next performed yeast two-hybrid screening and identified radixin, a member of the ERM (ezrin, radixin, moesin) protein family, as a potential binding partner of KIAA0319. ERM proteins are adapter molecules that link membrane proteins to actin cytoskeletone and involved in the regulation of cellular morphology and movement. Pull-down experiments indicated that radixin specifically bound to the intracellular region of KIAA0319. We are now trying to clarify the physiological significance of the interaction in primary cultured neurons, which may lead to better understanding of the molecular mechanisms involved in dyslexia.

KCC2 prioritizes leading processes to be primary dendrites

Developing neuronal networks generate various patterns of synchronized oscillatory activity that is believed to participate in circuit refinement. In the immature cortex and hippocampus, two major forms of the synchronized activity have been well documented, i.e., early network oscillations and giant depolarizing potentials. However, the neuronal activity patterns in other memory-relevant networks such as the amygdala still remain unclear. Using a functional multineuron calcium imaging technique that monitors the activity of thousands of cells, we revealed that synchronized waves propagated from the cortex to the lateral/basolateral amygdala in acute horizontal slice of developing rat. This cortico-amygdalar waves occasionally activated the central amygdala and terminated within the amygdala. The origin of the synchronized waves was likely located in the entorhinal and perirhinal cortex, which correspond to anatomically important networks that connect the hippocampus and amygdala with large parts of the neocortex. To the best of our knowledge, these findings demonstrate the first report showing the early amygdalar network oscillations.

Proper dendritic morphogenesis requires KCC2 expression in immature dentate granule cells

axons from the explant exhibited radial outgrowth into thematrix. However, the explants were co-cultured with the notochord explants, growing Cnt1-positive spinal axons were repelled by notochord-derived repulsive activity. This repulsive activity was abolished by the addition of either a monoclonal anti-Cnt1 antibody, chondroitin sulfate A or chondroitinase ABC to the culturemedium. An antibody that recognizes chondroitin sulfate A and C labeled immunohistochemically the notochord in embryo sections and the collagen gelmatrix around the cultured notochord explant. Addition of chondroitinase ABC into the culture eliminated the immunoreactivity in the gel matrix. These results suggest that the notochord-derived chondroitin sulfate proteoglycan acts as a repulsive signaling molecule that is recognized by Cnt1 on spinal sensory axons. This guidance mechanism would be involved in navigation of the growing sensory axons not to enter mid-body areas where the notochord exist, resulting in their subepidermal extension.

Axonal calcium sweep: Somatic remote control of axon elongation

In the developing cerebral neocortex, postmitotic neurons in the ventricular zone (VZ) do not directly migrate into the cortical plate (CP) but stay for some time in the subventricular zone (SVZ) with multipolar morphology. In this zone, they dynamically extend and retract multiple processes, moving as if they are seeking for some extracellular signals. However, what molecules control this ‘multipolar migration’ is still unknown. In this study, we identified receptor molecules that are preferentially expressed in the multipolar migration neurons in the SVZ. Although some of the ligand molecules for these receptors were not produced in the CP, these ligand proteins were detected in the intermediate zone or the VZ, facing to the SVZ. These coordinated distribution patterns of receptors and their ligands suggest that the behavior of multipolar migration neurons may be regulated by the adjacent structures through the ligand-receptor interactions.

GABA and glutamate regulate the differentiation of newborn hippocampal granule cells

Chondroitin sulfates (CSs), carbohydrate side chains of chondroitin sulfate proteoglycans, are involved in formation of retinotectal pathway in chick embryos, delimiting a border of the pathway and chronological sorting of the axons in the optic tract. CSs exhibit structural diversity with various disaccharide units combined; however, it has not been shown whether the CSs affect the growth cones directly or not. In addition, there have been few studies about the structural diversity and functional specificity. A method called the spot assay, examining growth of the axons around the spot border, was not suitable for the study about CSs. Here we established a method to elucidate effects of the CSs and their structural diversity and functional specificity. We investigate behaviors of the growth cones in contact with the polystyrene beads coated with chondroitin sulfate phosphatidylethanolamine.