Haruo Kasai

Structural basis of long-term potentiation in single dendritic spines

Dendritic spines of pyramidal neurons in the cerebral cortex undergo activity-dependent structural remodelling that has been proposed to be a cellular basis of learning and memory. How structural remodelling supports synaptic plasticity, such as long-term potentiation, and whether such plasticity is input-specific at the level of the individual spine has remained unknown. We investigated the structural basis of long-term potentiation using two-photon photolysis of caged glutamate at single spines of hippocampal CA1 pyramidal neurons. Here we show that repetitive quantum-like photorelease (uncaging) of glutamate induces a rapid and selective enlargement of stimulated spines that is transient in large mushroom spines but persistent in small spines. Spine enlargement is associated with an increase in AMPA-receptor-mediated currents at the stimulated synapse and is dependent on NMDA receptors, calmodulin and actin polymerization. Long-lasting spine enlargement also requires Ca2+/calmodulin-dependent protein kinase II. Our results thus indicate that spines individually follow Hebbs postulate for learning. They further suggest that small spines are preferential sites for long-term potentiation induction, whereas large spines might represent physical traces of long-term memory.

Dendritic spine geometry is critical for AMPA receptor expression in hippocampal CA1 pyramidal neurons

Dendritic spines serve as preferential sites of excitatory synaptic connections and are pleomorphic. To address the structure–function relationship of the dendritic spines, we used two-photon uncaging of glutamate to allow mapping of functional glutamate receptors at the level of the single synapse. Our analyses of the spines of CA1 pyramidal neurons reveal that AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)-type glutamate receptors are abundant (up to 150/spine) in mushroom spines but sparsely distributed in thin spines and filopodia. The latter may be serving as the structural substrates of the silent synapses that have been proposed to play roles in development and plasticity of synaptic transmission. Our data indicate that distribution of functional AMPA receptors is tightly correlated with spine geometry and that receptor activity is independently regulated at the level of single spines.

Protein Synthesis and Neurotrophin-Dependent Structural Plasticity of Single Dendritic Spines

Long-term potentiation (LTP) at glutamatergic synapses is considered to underlie learning and memory and is associated with the enlargement of dendritic spines. Because the consolidation of memory and LTP require protein synthesis, it is important to clarify how protein synthesis affects spine enlargement. In rat brain slices, the repetitive pairing of postsynaptic spikes and two-photon uncaging of glutamate at single spines (a spike-timing protocol) produced both immediate and gradual phases of spine enlargement in CA1 pyramidal neurons. The gradual enlargement was strongly dependent on protein synthesis and brain-derived neurotrophic factor (BDNF) action, often associated with spine twitching, and was induced specifically at the spines that were immediately enlarged by the synaptic stimulation. Thus, this spike-timing protocol is an efficient trigger for BDNF secretion and induces protein synthesis–dependent long-term enlargement at the level of single spines.

High-speed mapping of synaptic connectivity using photostimulation in Channelrhodopsin-2 transgenic mice

To permit rapid optical control of brain activity, we have engineered multiple lines of transgenic mice that express the light-activated cation channel Channelrhodopsin-2 (ChR2) in subsets of neurons. Illumination of ChR2-positive neurons in brain slices produced photocurrents that generated action potentials within milliseconds and with precisely timed latencies. The number of light-evoked action potentials could be controlled by varying either the amplitude or duration of illumination. Furthermore, the frequency of light-evoked action potentials could be precisely controlled up to 30 Hz. Photostimulation also could evoke synaptic transmission between neurons, and, by scanning with a small laser light spot, we were able to map the spatial distribution of synaptic circuits connecting neurons within living cerebral cortex. We conclude that ChR2 is a genetically based photostimulation technology that permits analysis of neural circuits with high spatial and temporal resolution in transgenic mammals.

Subcellular distribution of Ca2+ release channels underlying Ca2+ waves and oscillations in exocrine pancreas

Agonists trigger Ca2+ waves and oscillations in exocrine gland cells. Our confocal Ca2+ imaging revealed three distinct phases during the Ca2+ waves in the rat pancreatic acinar cell. Rises in Ca2+ concentration were initiated at a small trigger zone, or T zone, in the granular area; then, Ca2+ waves rapidly spread within the area and, at high agonist concentrations, propagated slowly toward the basal pole. Injection of inositol 1,4,5-trisphosphate (IP3) or Ca2+ from patch pipettes demonstrated the presence of high sensitivity IP3 receptors at the T zone, Ca(2+)-induced Ca2+ release channels in the granular area, and low sensitivity IP3 receptors in the basal area. The IP3 receptors at the T zone appeared to generate autonomous Ca2+ spikes and to initiate patterned Ca2+ oscillations. Thus, heterogeneous cytosolic localization of Ca2+ release channels plays a key role in Ca2+ waves and oscillations.

Spine-Neck Geometry Determines NMDA Receptor-Dependent Ca2+ Signaling in Dendrites

Increases in cytosolic Ca2+ concentration ([Ca2+]i) mediated by NMDA-sensitive glutamate receptors (NMDARs) are important for synaptic plasticity. We studied a wide variety of dendritic spines on rat CA1 pyramidal neurons in acute hippocampal slices. Two-photon uncaging and Ca2+ imaging revealed that NMDAR-mediated currents increased with spine-head volume and that even the smallest spines contained a significant number of NMDARs. The fate of Ca2+ that entered spine heads through NMDARs was governed by the shape (length and radius) of the spine neck. Larger spines had necks that permitted greater efflux of Ca2+ into the dendritic shaft, whereas smaller spines manifested a larger increase in [Ca2+]i within the spine compartment as a result of a smaller Ca2+ flux through the neck. Spine-neck geometry is thus an important determinant of spine Ca2+ signaling, allowing small spines to be the preferential sites for isolated induction of long-term potentiation.

Spatial dynamics of second messengers : IP3 and cAMP as long-range and associative messengers

Recent imaging experiments have revealed the distinct spatial dynamics of second-messenger actions. In general, actions of Ca2+ tend to be local, whereas those of other messengers such as inositol 1,4,5-trisphosphate (IP3) and cAMP are long range. In pancreatic acinar cells, IP3 generated at the base can diffuse across the cell and evoke a spatially confined Ca2+ signal in the apical pole, triggering enzyme and fluid secretion. Similar mechanisms might also operate in other cell types. We propose that the distinct dynamics of messengers might be relevant to neuronal function: IP3 and cAMP could convey signals over long distances along neurites, and serve as mediators for association and co-operation, for example, during learning.

Principles of Long-Term Dynamics of Dendritic Spines

Long-term potentiation of synapse strength requires enlargement of dendritic spines on cerebral pyramidal neurons. Long-term depression is linked to spine shrinkage. Indeed, spines are dynamic structures: they form, change their shapes and volumes, or can disappear in the space of hours. Do all such changes result from synaptic activity, or do some changes result from intrinsic processes? How do enlargement and shrinkage of spines relate to elimination and generation of spines, and how do these processes contribute to the stationary distribution of spine volumes? To answer these questions, we recorded the volumes of many individual spines daily for several days using two-photon imaging of CA1 pyramidal neurons in cultured slices of rat hippocampus between postnatal days 17 and 23. With normal synaptic transmission, spines often changed volume or were created or eliminated, thereby showing activity-dependent plasticity. However, we found that spines changed volume even after we blocked synaptic activity, reflecting a native instability of these small structures over the long term. Such “intrinsic fluctuations” showed unique dependence on spine volume. A mathematical model constructed from these data and the theory of random fluctuations explains population behaviors of spines, such as rates of elimination and generation, stationary distribution of volumes, and the long-term persistence of large spines. Our study finds that generation and elimination of spines are more prevalent than previously believed, and spine volume shows significant correlation with its age and life expectancy. The population dynamics of spines also predict key psychological features of memory.

Characterization of two kinds of high-voltage-activated Ca-channel currents in chick sensory neurons

High-voltage-activated (HVA) Ca-channel currents in chick sensory neurons were characterized by dihydropyridine compounds (DHPs) and ω-conotoxin GVIA (ωCTX) using patch-clamp methods. In single-channel recordings, two HVA-currents were identified by their single-channel conductances, 13 pS and 25 pS in 110 mM BaCl2. DHPs selectively affected the large-conductance channel. ωCTX (5 μM), on the other hand, irreversibly eliminated only the small-conductance channel, while the large-conductance channel was either unaffected or only transiently blocked. In whole-cell recordings the macroscopic HVA-current was completely and irreversibly blocked by ωCTX but insensitive to DHPs in 60% of the cells. This current presumably was carried by the 13 pS channel. In the remaining cells, a part of the HVA-current (10%, SD=11%) was either unaffected or transiently blocked by ωCTX and was sensitive to DHPs. This current presumably was carried by the 25 pS channel. Inactivation of both macroscopic current component was incomplete during a 150 ms long depolarization. Our data suggest that the HVA-currents in chick sensory neurons are carried by two distinct Ca-channels that are differentially affected by ωCTX and DHPs.

PANCREATIC BETA -CELL-SPECIFIC TARGETED DISRUPTION OF GLUCOKINASE GENE : DIABETES MELLITUS DUE TO DEFECTIVE INSULIN SECRETION TO GLUCOSE

Mice carrying a null mutation in the glucokinase (GK) gene in pancreatic β-cells, but not in the liver, were generated by disrupting the β-cell-specific exon. Heterozygous mutant mice showed early-onset mild diabetes due to impaired insulin-secretory response to glucose. Homozygotes showed severe diabetes shortly after birth and died within a week. GK-deficient islets isolated from homozygotes showed defective insulin secretion in response to glucose, while they responded to other secretagogues: almost normally to arginine and to some extent to sulfonylureas. These data provide the first direct proof that GK serves as a glucose sensor molecule for insulin secretion and plays a pivotal role in glucose homeostasis. GK-deficient mice serve as an animal model of the insulin-secretory defect in human non-insulin-dependent diabetes mellitus.