Hajime Takano

Synaptic Islands Defined by the Territory of a Single Astrocyte

In the mammalian brain, astrocytes modulate neuronal function, in part, by synchronizing neuronal firing and coordinating synaptic networks. Little, however, is known about how this is accomplished from a structural standpoint. To investigate the structural basis of astrocyte-mediated neuronal synchrony and synaptic coordination, the three-dimensional relationships between cortical astrocytes and neurons was investigated. Using a transgenic and viral approach to label astrocytes with enhanced green fluorescent protein, we performed a three-dimensional reconstruction of astrocytes from tissue sections or live animals in vivo. We found that cortical astrocytes occupy nonoverlapping territories similar to those described in the hippocampus. Using immunofluorescence labeling of neuronal somata, a single astrocyte enwraps on average four neuronal somata with an upper limit of eight. Single-neuron dye-fills allowed us to estimate that one astrocyte contacts 300–600 neuronal dendrites. Together with the recent findings showing that glial Ca2+ signaling is restricted to individual astrocytes in vivo, and that Ca2+ signaling leads to gliotransmission, we propose the concept of functional islands of synapses in which groups of synapses confined within the boundaries of an individual astrocyte are modulated by the gliotransmitter environment controlled by that astrocyte. Our description offers a new structurally based conceptual framework to evaluate functional data involving interactions between neurons and astrocytes in the mammalian brain.

Selective induction of astrocytic gliosis generates deficits in neuronal inhibition

Reactive astrocytosis develops in many neurologic diseases, including epilepsy. Astrocytotic contributions to pathophysiology are poorly understood. Studies examining this are confounded by comorbidities accompanying reactive astrocytosis. We found that high-titer transduction of astrocytes with enhanced green fluorescent protein (eGFP) via adeno-associated virus induced reactive astrocytosis without altering the intrinsic properties or anatomy of neighboring neurons. We examined the consequences of selective astrocytosis induction on synaptic transmission in mouse CA1 pyramidal neurons. Neurons near eGFP-labeled reactive astrocytes had reduced inhibitory, but not excitatory, synaptic currents. This inhibitory postsynaptic current (IPSC) erosion resulted from a failure of the astrocytic glutamate-glutamine cycle. Reactive astrocytes downregulated expression of glutamine synthetase. Blockade of this enzyme normally induces rapid synaptic GABA depletion. In astrocytotic regions, residual inhibition lost sensitivity to glutamine synthetase blockade, whereas exogenous glutamine administration enhanced IPSCs. Astrocytosis-mediated deficits in inhibition triggered glutamine-reversible hyperexcitability in hippocampal circuits. Thus, reactive astrocytosis could generate local synaptic perturbations, leading to broader functional deficits associated with neurologic disease.

Random migration precedes stable target cell interactions of tumor-infiltrating T cells

The tumor microenvironment is composed of an intricate mixture of tumor and host-derived cells that engage in a continuous interplay. T cells are particularly important in this context as they may recognize tumor-associated antigens and induce tumor regression. However, the precise identity of cells targeted by tumor-infiltrating T lymphocytes (TILs) as well as the kinetics and anatomy of TIL-target cell interactions within tumors are incompletely understood. Furthermore, the spatiotemporal conditions of TIL locomotion through the tumor stroma, as a prerequisite for establishing contact with target cells, have not been analyzed. These shortcomings limit the rational design of immunotherapeutic strategies that aim to overcome tumor-immune evasion. We have used two-photon microscopy to determine, in a dynamic manner, the requirements leading to tumor regression by TILs. Key observations were that TILs migrated randomly throughout the tumor microenvironment and that, in the absence of cognate antigen, they were incapable of sustaining active migration. Furthermore, TILs in regressing tumors formed long-lasting (≥30 min), cognate antigen–dependent contacts with tumor cells. Finally, TILs physically interacted with macrophages, suggesting tumor antigen cross-presentation by these cells. Our results demonstrate that recognition of cognate antigen within tumors is a critical determinant of optimal TIL migration and target cell interactions, and argue against TIL guidance by long-range chemokine gradients.

Endogenous nonneuronal modulators of synaptic transmission control cortical slow oscillations in vivo

Gliotransmission, the release of molecules from astrocytes, regulates neuronal excitability and synaptic transmission in situ. Whether this process affects neuronal network activity in vivo is not known. Using a combination of astrocyte-specific molecular genetics, with in vivo electrophysiology and pharmacology, we determined that gliotransmission modulates cortical slow oscillations, a rhythm characterizing nonrapid eye movement sleep. Inhibition of gliotransmission by the expression of a dominant negative SNARE domain in astrocytes affected cortical slow oscillations, reducing the duration of neuronal depolarizations and causing prolonged hyperpolarizations. These network effects result from the astrocytic modulation of intracortical synaptic transmission at two sites: a hypofunction of postsynaptic NMDA receptors, and by reducing extracellular adenosine, a loss of tonic A1 receptor-mediated inhibition. These results demonstrate that rhythmic brain activity is generated by the coordinated action of the neuronal and glial networks.

Transparent and flexible low noise graphene electrodes for simultaneous electrophysiology and neuroimaging

Calcium imaging is a versatile experimental approach capable of resolving single neurons with single-cell spatial resolution in the brain. Electrophysiological recordings provide high temporal, but limited spatial resolution, because of the geometrical inaccessibility of the brain. An approach that integrates the advantages of both techniques could provide new insights into functions of neural circuits. Here, we report a transparent, flexible neural electrode technology based on graphene, which enables simultaneous optical imaging and electrophysiological recording. We demonstrate that hippocampal slices can be imaged through transparent graphene electrodes by both confocal and two-photon microscopy without causing any light-induced artefacts in the electrical recordings. Graphene electrodes record high-frequency bursting activity and slow synaptic potentials that are hard to resolve by multicellular calcium imaging. This transparent electrode technology may pave the way for high spatio-temporal resolution electro-optic mapping of the dynamic neuronal activity.

Friction force microscopy of mixed Langmuir-Blodgett films

Thin films of mixtures of fluorocarbons and hydrocarbons are investigated by friction force microscopy. For a mixture of equal molar parts of arachidic acid and partially fluorinated carboxylic acid, phase separation manifests itself as circular domains. By shortening the length of the hydrocarbon chain, the disorder of the system is increased and phase separation takes on a fractal geometry. The frictional forces are found to be material specific and distinguish between domains of fluorocarbons and hydrocarbons. With higher normal forces hydrocarbon particles are easily sheared away while the fluorocarbon region remains unbroken. The results are discussed in relation to the phenomenon of boundary lubrication.

Neuronal Activity and Glutamate Uptake Decrease Mitochondrial Mobility in Astrocytes and Position Mitochondria Near Glutamate Transporters

Within neurons, mitochondria are nonuniformly distributed and are retained at sites of high activity and metabolic demand. Glutamate transport and the concomitant activation of the Na+/K+-ATPase represent a substantial energetic demand on astrocytes. We hypothesized that mitochondrial mobility within astrocytic processes might be regulated by neuronal activity and glutamate transport. We imaged organotypic hippocampal slice cultures of rat, in which astrocytes maintain their highly branched morphologies and express glutamate transporters. Using time-lapse confocal microscopy, the mobility of mitochondria within individual astrocytic processes and neuronal dendrites was tracked. Within neurons, a greater percentage of mitochondria were mobile than in astrocytes. Furthermore, they moved faster and farther than in astrocytes. Inhibiting neuronal activity with tetrodotoxin (TTX) increased the percentage of mobile mitochondria in astrocytes. Mitochondrial movement in astrocytes was inhibited by vinblastine and cytochalasin D, demonstrating that this mobility depends on both the microtubule and actin cytoskeletons. Inhibition of glutamate transport tripled the percentage of mobile mitochondria in astrocytes. Conversely, application of the transporter substrate d-aspartate reversed the TTX-induced increase in the percentage of mobile mitochondria. Inhibition of reversed Na+/Ca2+ exchange also increased the percentage of mitochondria that were mobile. Last, we demonstrated that neuronal activity increases the probability that mitochondria appose GLT-1 particles within astrocyte processes, without changing the proximity of GLT-1 particles to VGLUT1. These results imply that neuronal activity and the resulting clearance of glutamate by astrocytes regulate the movement of astrocytic mitochondria and suggest a mechanism by which glutamate transporters might retain mitochondria at sites of glutamate uptake.

Axonal α7 nicotinic ACh receptors modulate presynaptic NMDA receptor expression and structural plasticity of glutamatergic presynaptic boutons

In association with NMDA receptors (NMDARs), neuronal α7 nicotinic ACh receptors (nAChRs) have been implicated in neuronal plasticity as well as neurodevelopmental, neurological, and psychiatric disorders. However, the role of presynaptic NMDARs and their interaction with α7 nAChRs in these physiological and pathophysiological events remains unknown. Here we report that axonal α7 nAChRs modulate presynaptic NMDAR expression and structural plasticity of glutamatergic presynaptic boutons during early synaptic development. Chronic inactivation of α7 nAChRs markedly increased cell surface NMDAR expression as well as the number and size of glutamatergic axonal varicosities in cortical cultures. These boutons contained presynaptic NMDARs and α7 nAChRs, and recordings from outside-out pulled patches of enlarged presynaptic boutons identified functional NMDAR-mediated currents. Multiphoton imaging of presynaptic NMDAR-mediated calcium transients demonstrated significantly larger responses in these enlarged boutons, suggesting enhanced presynaptic NMDAR function that could lead to increased glutamate release. Moreover, whole-cell patch clamp showed a significant increase in synaptic charge mediated by NMDAR miniature EPSCs but no alteration in the frequency of AMPAR miniature EPSCs, suggesting the selective enhancement of postsynaptically silent synapses upon inactivation of α7 nAChRs. Taken together, these findings indicate that axonal α7 nAChRs modulate presynaptic NMDAR expression and presynaptic and postsynaptic maturation of glutamatergic synapses, and implicate presynaptic α7 nAChR/NMDAR interactions in synaptic development and plasticity.

Axon Regeneration in the Absence of Growth Cones: Acceleration by Cyclic AMP

Regenerative failure of spinal axons is commonly ascribed to signaling of F‐actin depolymerization and growth cone collapse by molecules such as the myelin‐associated growth inhibitors. cAMP is thought to promote regeneration at least in part by neutralizing this effect, either by direct action in the growth cone or indirectly by transcriptional mechanisms. In vivo evidence for this is based mainly on partial lesion studies in which it is sometimes difficult to distinguish regeneration of injured axons from collateral sprouting by uninjured axons. Moreover, previous observations on fixed lamprey central nervous system (CNS) suggested that regeneration may not involve growth cones. To distinguish actively growing axons from static or retracting ones, fluorescently labeled large reticulospinal axons were imaged in the living, transected lamprey cord with and without application of cAMP analogs and then studied by 2‐photon microscopy. Axon tip movements over 2–48‐hour intervals indicated: 1) regeneration was intermittent; 2) cAMP decreased initial axon retraction and increased subsequent regeneration up to 11‐fold; 3) the increase in regeneration was due to an increase in velocity of axon growth, but not in the time spent in forward movement; 4) tips of actively regenerating axons were more sharply contoured than static tips but no filopodia or lamellipodia were observed, even in db‐cAMP; and 5) during active growth, axon tips contained vesicle‐like inclusions and were highly immunoreactive for neurofilaments. Staining for F‐actin and microtubules was variable and F‐actin was not concentrated at the leading edge. Thus, cAMP accelerates regeneration of lamprey spinal axons without inducing formation of growth cones. J. Comp. Neurol. 515:295–312, 2009.

Suppression of InsP3 Receptor-Mediated Ca2+ Signaling Alleviates Mutant Presenilin-Linked Familial Alzheimer's Disease Pathogenesis

Exaggerated intracellular Ca2+ signaling is a robust proximal phenotype observed in cells expressing familial Alzheimers disease (FAD)-causing mutant presenilins (PSs). The mechanisms that underlie this phenotype are controversial and their in vivo relevance for AD pathogenesis is unknown. Here, we used a genetic approach to identify the mechanisms involved and to evaluate their role in the etiology of AD in two FAD mouse models. Genetic reduction of the type 1 inositol trisphosphate receptor (InsP3R1) by 50% normalized exaggerated Ca2+ signaling observed in cortical and hippocampal neurons in both animal models. In PS1M146V knock-in mice, reduced InsP3R1 expression restored normal ryanodine receptor and cAMP response element-binding protein (CREB)-dependent gene expression and rescued aberrant hippocampal long-term potentiation (LTP). In 3xTg mice, reduced InsP3R1 expression profoundly attenuated amyloid β accumulation and tau hyperphosphorylation and rescued hippocampal LTP and memory deficits. These results indicate that exaggerated Ca2+ signaling, which is associated with FAD PS, is mediated by InsP3R and contributes to disease pathogenesis in vivo. Targeting the InsP3 signaling pathway could be considered a potential therapeutic strategy for patients harboring mutations in PS linked to AD.