Ryuta Koyama

Brain-Derived Neurotrophic Factor Induces Hyperexcitable Reentrant Circuits in the Dentate Gyrus

Aberrant sprouting and synaptic reorganization of the mossy fiber (MF) axons are commonly found in the hippocampus of temporal lobe epilepsy patients and result in the formation of excitatory feedback loops in the dentate gyrus, a putative cellular basis for recurrent epileptic seizures. Using ex vivo hippocampal cultures, we show that prolonged hyperactivity induces MF sprouting and the resultant network reorganizations and that brain-derived neurotrophic factor (BDNF) is necessary and sufficient to evoke these pathogenic plasticities. Hyperexcitation induced an upregulation of BDNF protein expression in the MF pathway, an effect mediated by L-type Ca2+ channels. The neurotrophin receptor tyrosine kinase (Trk)B inhibitor K252a or function-blocking anti-BDNF antibody prevented hyperactivity-induced MF sprouting. Even under blockade of neural activity, local application of BDNF to the hilus, but not other subregions, was capable of initiating MF axonal remodeling, eventually leading to dentate hyperexcitability. Transfecting granule cells with dominant-negative TrkB prevented axonal branching. Thus, excessive activation of L-type Ca2+ channels causes granule cells to express BDNF, and extracellularly released BDNF stimulates TrkB receptors present on the hilar segment of the MFs to induce axonal branching, which may establish hyperexcitable dentate circuits.

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.

To BDNF or Not to BDNF: That Is the Epileptic Hippocampus:

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, has drawn much attention as a potential therapeutic target for temporal lobe epilepsy (TLE). TLE seizures are produced by synchronized hyperactivity of neuron populations due to the disruption of a balance between excitatory and inhibitory synaptic transmissions. In epileptogenesis-related brain areas, including the hippocampus, BDNF is up-regulated in the course of the development of epilepsy and induces a collapse of balanced excitation and inhibition, eventually exerting its epileptogenic effects. On the other hand, several reports demonstrate that intrahippocampal infusion of BDNF can attenuate (or retard) the development of epilepsy. This antiepileptogenic effect seems to be mediated mainly by an increase in the expression of neuropeptide Y. These contrasting effects of BDNF have prevented us from concluding whether inhibition or enhancement of BDNF signaling finally achieves the prevention of TLE. To address this question, it is essential to evaluate how BDNF changes its influences depending on conditions, for example, cell specificity, neural networks, and expression timing and loci. In this article, the authors review BDNF-induced acute and long-lasting changes seen in epileptic circuits from the anatomical and functional points of view.

Mossy Fiber Sprouting as a Potential Therapeutic Target for Epilepsy

Hippocampal mossy fibers, axons of dentate granule cells, converge in the dentate hilus and run through a narrow area called the stratum lucidum to synapse with hilar and CA3 neurons. In the hippocampal formation of temporal lobe epilepsy patients, however, this stereotyped pattern of projection is often collapsed; the mossy fibers branch out of the dentate hilus and abnormally innervate the dentate inner molecular layer, a phenomenon that is termed mossy fiber sprouting. Experimental studies have replicated this sprouting in animal models of temporal lobe epilepsy, including kindling and pharmacological treatment with convulsants. Because these axon collaterals form recurrent excitatory inputs into dendrites of granule cells, the circuit reorganization is assumed to cause epileptiform activity in the hippocampus, whereas some recent studies indicate that the sprouting is not necessarily associated with early-life seizures. Here we review the mechanisms of mossy fiber sprouting and consider its potential contribution to epileptogenesis. Based on recent findings, we propose that the sprouting can be regarded as a result of disruption of the molecular mechanisms underlying the axon guidance. We finally focus on the possibility that prevention of the abnormal sprouting might be a new strategy for medical treatment with temporal lobe epilepsy.

Neonatally born granule cells numerically dominate adult mice dentate gyrus.

Hippocampal granule cells (GCs) are continuously generated in the subgranular zone of the dentate gyrus (DG) and functionally incorporated to dentate neural circuits even in adulthood. This raises a question about the fate of neonatally born GCs in adult DG. Do they exist until adulthood or are they largely superseded by adult-born GCs? To investigate this question, we examined the contributions of postnatally born GCs to the adult mouse DG. C57BL/6 mice were grouped in three different postnatal (P) ages (group 1: P0, group 2: P7, and group 3: P35) and received a daily bromodeoxyuridine (BrdU) injection for three consecutive days (P0/1/2, P7/8/9, and P35/36/37, respectively) to label dividing cells. At 6 months old, hippocampal sections were prepared from the animals and immunostained with anti-BrdU antibody and an antibody against the homeobox prospero-like protein Prox1, a marker of GCs. We defined BrdU- and Prox1-double positive cells as newborn GCs and analyzed their density and distribution in the granule cell layer (gcl), revealing that newborn GCs of each group still existed 6 months after BrdU injections and that the density of GCs born during P0-2 (group 1) was significantly higher compared with the other groups. Although the density of newborn GCs in the each group did not differ between male and female, the radial distribution of them in gcl showed some differences, that is, male newborn GCs localized toward the molecular layer compared with female ones in group 1, while to the hilus in group 2. These results suggest that GCs born in early postnatal days numerically dominate adult DG and that there exist sex differences in GC localizations which depend on the time when they were born.

Prenatal Stress Inhibits Neuronal Maturation through Downregulation of Mineralocorticoid Receptors

Prenatal stress (PS) increases the risk of depressive disorders in adult offspring. The pathophysiology of depressive disorders has been linked to hippocampal dysfunction; however, whether and how PS attenuates the development and function of hippocampal networks remains unknown. Using a rat model of PS, in which pregnant mothers receive daily restraint stress during late gestation and their offspring exhibit depressive-like behavior later in life, we show that PS impairs the morphological and functional maturation of hippocampal granule cells in adult offspring via the downregulated expression of mineralocorticoid receptors. PS reduced the dendritic complexity and spine density of neonatal-generated granule cells, which persists into adulthood. These granule cells exhibited depressed synaptic responses to stimulation of the medial perforant path. We further revealed that the expression of mineralocorticoid receptors, which we found is necessary for proper dendritic maturation in this study, was significantly downregulated in granule cells after PS. These results suggest that PS-induced downregulation of mineralocorticoid receptors attenuates neuronal maturation, which results in dysfunction of neuronal network in adulthood.

AMP-activated protein kinase mediates activity-dependent axon branching by recruiting mitochondria to axon

During development, axons are guided to their target areas and provide local branching. Spatiotemporal regulation of axon branching is crucial for the establishment of functional connections between appropriate pre‐ and postsynaptic neurons. Common understanding has been that neuronal activity contributes to the proper axon branching; however, intracellular mechanisms that underlie activity‐dependent axon branching remain elusive. Here, we show, using primary cultures of the dentate granule cells, that neuronal depolarization‐induced rebalance of mitochondrial motility between anterograde versus retrograde transport underlies the proper formation of axonal branches. We found that the depolarization‐induced branch formation was blocked by the uncoupler p‐trifluoromethoxyphenylhydrazone, which suggests that mitochondria‐derived ATP mediates the observed phenomena. Real‐time analysis of mitochondrial movement defined the molecular mechanisms by showing that the pharmacological activation of AMP‐activated protein kinase (AMPK) after depolarization increased anterograde transport of mitochondria into axons. Simultaneous imaging of axonal morphology and mitochondrial distribution revealed that mitochondrial localization preceded the emergence of axonal branches. Moreover, the higher probability of mitochondrial localization was correlated with the longer lifetime of axon branches. We qualitatively confirmed that neuronal ATP levels decreased immediately after depolarization and found that the phosphorylated form of AMPK was increased. Thus, this study identifies a novel role for AMPK in the transport of axonal mitochondria that underlie the neuronal activity‐dependent formation of axon branches.

Microglia in the pathogenesis of autism spectrum disorders

Proper synaptic pruning is essential for the development of functional neural circuits. Impairments in synaptic pruning disrupt the excitatory versus inhibitory balance (E/I balance) of synapses, which may cause neurodevelopmental disorders such as autism spectrum disorder (ASD). Recent studies have determined molecular mechanisms by which microglia, the brains resident immune cells, engulf inappropriate and less active synapses. Thus, microglial dysfunction may be involved in the pathogenesis of ASD through attenuated or excess synaptic pruning. In this review, we discuss recent animal and human studies that report an E/I imbalance and the characteristics of microglia in ASD. We will further discuss whether and how synaptic pruning by microglia is involved in the pathogenesis of ASD.

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.

Group II metabotropic glutamate receptor activation is required for normal hippocampal mossy fibre development in the rat

Glutamate is the main neurotransmitter at hippocampal mossy fibre (MF) terminals. Because neurotransmitters have been proposed as regulating factors of neural network formation and neurite morphogenesis in the developing CNS, we examined the possible contribution of glutamate to MF pathfinding. Entorhino‐hippocampal slices prepared from early postnatal rats were cultivated in the presence of glutamate receptor antagonists. Timm histochemical staining revealed that pharmacological blockade of metabotropic glutamate receptors (mGluR), but not of ionotropic glutamate receptors, induced abnormal outgrowth of the MFs. When slices were cultured in the presence of mGluR antagonists, DiI‐labelled MF axons displayed a great degree of defasciculation, and MF‐mediated EPSPs in the CA3 pyramidal cells were altered. Similar results were obtained for a selective antagonist of group II mGluR, but not of group I or III mGluR. Glutamate is, therefore, likely to regulate MF outgrowth via activation of group II mGluR. The present study may provide a novel role of glutamate in hippocampal development.