山下 貴之

<title>Abstract</title>Scintillators emit visible luminescence when irradiated with X-rays. Given the unlimited tissue penetration of X-rays, the employment of scintillators could enable remote optogenetic control of neural functions at any depth of the brain. Here we show that a yellow-emitting inorganic scintillator, Ce-doped Gd₃(Al,Ga)₅O₁₂ (Ce:GAGG), can effectively activate red-shifted excitatory and inhibitory opsins, ChRmine and GtACR1, respectively. Using injectable Ce:GAGG microparticles, we successfully activated and inhibited midbrain dopamine neurons in freely moving mice by X-ray irradiation, producing bidirectional modulation of place preference behavior. Ce:GAGG microparticles are non-cytotoxic and biocompatible, allowing for chronic implantation. Pulsed X-ray irradiation at a clinical dose level is sufficient to elicit behavioral changes without reducing the number of radiosensitive cells in the brain and bone marrow. Thus, scintillator-mediated optogenetics enables minimally invasive, wireless control of cellular functions at any tissue depth in living animals, expanding X-ray applications to functional studies of biology and medicine.

Excitatory projection neurons of the neocortex are thought to play important roles in perceptual and cognitive functions of the brain by directly connecting diverse cortical and subcortical areas. However, many aspects of the anatomical organization of these inter-areal connections are unknown. Here, we studied long-range axonal projections of excitatory layer 2/3 neurons with cell bodies located in mouse primary somatosensory barrel cortex (wS1). As a population, these neurons densely projected to secondary whisker somatosensory cortex (wS2) and primary/secondary whisker motor cortex (wM1/2), with additional axon in the dysgranular zone surrounding the barrel field, perirhinal temporal association cortex and striatum. In three-dimensional reconstructions of 6 individual wS2-projecting neurons and 9 individual wM1/2-projecting neurons, we found that both classes of neurons had extensive local axon in layers 2/3 and 5 of wS1. Neurons projecting to wS2 did not send axon to wM1/2, whereas a small subset of wM1/2-projecting neurons had relatively weak projections to wS2. A small fraction of projection neurons solely targeted wS2 or wM1/2. However, axon collaterals from wS2-projecting and wM1/2-projecting neurons were typically also found in subsets of various additional areas, including the dysgranular zone, perirhinal temporal association cortex and striatum. Our data suggest extensive diversity in the axonal targets selected by individual nearby cortical long-range projection neurons with somata located in layer 2/3 of wS1.

A substantial portion of our sensory experience happens during active behaviors such as walking around or paying attention. How do sensory systems work during such behaviors? Neural processing in sensory systems can be shaped by behavior in multiple ways ranging from a modulation of responsiveness or sharpening of tuning to a dynamic change of response properties or functional connectivity. Here, we review recent findings on the modulation of sensory processing during active behaviors in different systems: insect vision, rodent thalamus, and rodent sensory cortices. We discuss the circuit-level mechanisms that might lead to these modulations and their potential role in sensory function. Finally, we highlight the open questions and future perspectives of this exciting new field.

The lateral hypothalamic area (LHA) of the diencephalon is crucially involved in controlling instinctive behavior such as sleep -wake cycle and feeding behavior. LHA is a heterogeneous structure that contains spatially intermingled, genetically distinct cell populations. Among LHA neurons, orexin/hypocretin (OX) neuron is the key cell type that promotes waking, and specific loss of OX neurons results in narcolepsy. Melanin-concentrating hormone (MCH) containing neurons are known to be active during rapid eye movement (REM) sleep and stimulation of these neurons promotes REM sleep. Here we review the classical and more recent findings in this field and discuss the molecular and cellular network organization of LHA neurons that could ultimately regulate the switch between wakefulness and general states of sleep.

Goal-directed behavior involves distributed neuronal circuits in the mammalian brain, including diverse regions of neocortex. However, the cellular basis of long-range cortico-cortical signaling during goal-directed behavior is poorly understood. Here, we recorded membrane potential of excitatory layer 2/3 pyramidal neurons in primary somatosensory barrel cortex (S1) projecting to either primary motor cortex (M1) or secondary somatosensory cortex (S2) during a whisker detection task, in which thirsty mice learn to lick for water reward in response to a whisker deflection. Whisker stimulation in 'Good performer' mice, but not 'Naive' mice, evoked long-lasting biphasic depolarization correlated with task performance in S2-projecting (S2-p) neurons, but not M1-projecting (M1-p) neurons. Furthermore, S2-p neurons, but not M1-p neurons, became excited during spontaneous unrewarded licking in 'Good performer' mice, but not in 'Naive' mice. Thus, a learning-induced, projection-specific signal from S1 to S2 may contribute to goal-directed sensorimotor transformation of whisker sensation into licking motor output.

Non-invasive remote control technologies designed to manipulate neural functions have been long-awaited for the comprehensive and quantitative understanding of neuronal network in the brain as well as for the therapy of neurological disorders. Recently, it has become possible for the neuronal activity to be optically manipulated using biological photo-reactive molecules such as channelrhodopsin (ChR)-2. However, ChR2 and its relatives are mostly reactive to visible light, which does not effectively penetrate through biological tissues. In contrast, near-infrared (NIR) light (650-1450 nm) penetrates deep into the tissues because biological systems are almost transparent to light within this so-called 'imaging window'. Here we used lanthanide nanoparticles (LNPs), composed of rare-earth elements, as luminous bodies to activate ChRs since they absorb low-energy NIR light to emit high-energy visible light (up-conversion). Here, we created a new type of optogenetic system which consists of the donor LNPs and the acceptor ChRs. The NIR laser irradiation emitted visible light from LNPs, then induced the photo-reactive responses in the near-by cells that expressed ChRs. However, there remains room for large improvements in the energy efficiency of the LNP-ChR system.

Primary sensory cortex discriminates incoming sensory information and generates multiple processing streams toward other cortical areas. However, the underlying cellular mechanisms remain unknown. Here, by making whole-cell recordings in primary somatosensory barrel cortex (Si) of behaving mice, we show that S1 neurons projecting to primary motor cortex (M1) and those projecting to secondary somatosensory cortex (S2) have distinct intrinsic membrane properties and exhibit markedly different membrane potential dynamics during behavior. Passive tactile stimulation evoked faster and larger postsynaptic potentials (PSPs) in M1-projecting neurons, rapidly driving phasic action potential firing, well-suited for stimulus detection. Repetitive active touch evoked strongly depressing PSPs and only transient firing in M1-projecting neurons. In contrast, PSP summation allowed S2-projecting neurons to robustly signal sensory information accumulated during repetitive touch, useful for encoding object features. Thus, target-specific transformation of sensory-evoked synaptic potentials by Si projection neurons generates functionally distinct output signals for sensorimotor coordination and sensory perception.

Action potentials, when arriving at presynaptic terminals, elicit Ca2+ influx through voltage-gated Ca2+ channels. Intracellular [Ca2+] elevation around the channels subsequently triggers synaptic vesicle exocytosis and also induces various protein reactions that regulate vesicle endocytosis and recycling to provide for long-term sustainability of synaptic transmission. Recent studies using membrane capacitance measurements, as well as high-resolution optical imaging, have revealed that the dominant type of synaptic vesicle endocytosis at central nervous system synapses is mediated by clathrin and dynamin. Furthermore. Ca2+-dependent mechanisms regulating endocytosis may operate in different ways depending on the distance from Ca2+ channels: (1) intracellular Ca2+ in the immediate vicinity of a Ca2+ channel plays an essential role in triggering endocytosis, and (2) intracellular Ca2+ traveling far from the channels has a modulatory effect on endocytosis at the periactive zone. Here. I integrate the latest progress in this field to propose a compartmental model for regulation of vesicle endocytosis at synapses and discuss the possible roles of presynaptic Ca2+-binding proteins including calmodulin, calcineurin and synaptotagmin. (c) 2012 Elsevier Ireland Ltd and the Japan Neuroscience Society. All rights reserved.

Ca2+ is thought to be essential for the exocytosis and endocytosis of synaptic vesicles. However, the manner in which Ca2+ coordinates these processes remains unclear, particularly at mature synapses. Using membrane capacitance measurements from calyx of Held nerve terminals in rats, we found that vesicle endocytosis is initiated primarily in Ca2+ nanodomains around Ca2+ channels, where exocytosis is triggered. Bulk Ca2+ outside of the domain could also be involved in endocytosis at immature synapses, although only after extensive exocytosis at more mature synapses. This bulk Ca2+-dependent endocytosis required calmodulin and calcineurin activation at immature synapses, but not at more mature synapses. Similarly, GTP-independent endocytosis, which occurred after extensive exocytosis at immature synapses, became negligible after maturation. We propose that nanodomain Ca2+ simultaneously triggers exocytosis and endocytosis of synaptic vesicles and that the molecular mechanisms underlying Ca2+-dependent endocytosis undergo major developmental changes at this fast central synapse.

The synaptic protein interaction (synprint) site of the voltage-gated Ca2+ channel (VGCC)alpha 1 subunit can interact with proteins involved in exocytosis, and it is therefore thought to be essential for exocytosis of synaptic vesicles. Here we report that the synprint site can also directly bind the mu subunit of AP-2, an adaptor protein for clathrin-mediated endocytosis, in competition with the synaptotagmin 1 (Syt 1) C2B domain. In brain lysates, the AP-2-synprint interaction occurred over a wide range of Ca2+ concentrations but was inhibited at high Ca2+ concentrations, in which Syt 1 interacted with synprint site. At the calyx of Held synapse in rat brainstem slices, direct presynaptic loading of the synprint fragment peptide blocked endocytic, but not exocytic, membrane capacitance changes. We propose that the VGCC synprint site is involved in synaptic vesicle endocytosis, rather than exocytosis, in the nerve terminal, via Ca2+-dependent interactions with AP-2 and Syt.

At central glutamatergic synapses, neurotransmitter often saturates postsynaptic AMPA receptors (AMPARs), thereby restricting the dynamic range of synaptic efficacy. Here, using simultaneous pre- and postsynaptic whole-cell recordings, at the calyx of Held synapse of immature rats, we have investigated the mechanism by which transmitter glutamate saturates postsynaptic AMPARs. When we loaded l-glutamate (1-100 mm) into presynaptic terminals, the quantal EPSC (qEPSC) amplitude changed in a concentration-dependent manner. At physiological temperature (36-37 degrees C), the qEPSC amplitude increased when intraterminal l-glutamate concentration was elevated from 1 mm to 10 mm, but it reached a plateau at 10 mm. This plateau persisted after bath-application of the low affinity AMPAR antagonist kynurenate, suggesting that it was caused by saturation of vesicular filling with glutamate rather than by saturation of postsynaptic AMPARs. In contrast to qEPSCs, action potential-evoked EPSCs remained unchanged by increasing intraterminal l-glutamate from 1 mm to 100 mm, even at room temperature, indicating that multi-quantal glutamate saturated postsynaptic AMPARs. This saturation could be relieved by blocking AMPAR desensitization using cyclothiazide (100 mu m). The concentration of ambient glutamate in the slice, estimated from NMDA receptor current fluctuations, was 55 nm; this was far below the concentration required for AMPAR desensitization. We conclude that rapid AMPAR desensitization, caused by glutamate released from multiple vesicles during synaptic transmission, underlies postsynaptic AMPAR saturation at this immature calyceal synapse before the onset of hearing.

Ca2+-binding to calmodulin (CaM) causes facilitation and/or inactivation of recombinant Ca2+ channels. At the rat calyx of Held, before hearing onset, presynaptic Ca2+ currents (I-pCa) undergo Ca2+/CaM-dependent inactivation during repetitive activation at around 1 Hz, implying that this may be a major cause of short-term synaptic depression. However, it remains open whether the Ca2+/CaM-dependent inactivation of I-pCa persists in more mature animals. To address this question, we tested the effect of CaM inhibitors on the activity-dependent modulation of I-pCa in calyces, before (postnatal day (P) 7-9) and after (P13-15) hearing onset. Our results indicate that the CaM-dependent I-pCa inactivation during low-frequency stimulation, and the ensuing synaptic depression, occur only at calyces in the prehearing period. However, CaM immunoreactivity in P8 and P14 calyces was equally strong. Even at P13-15, high frequency stimulation (200-500 Hz) could induce I-pCa inactivation, which was attenuated by EGTA (10 mM) or a CaM inhibitor peptide loaded into the terminal. Furthermore, the CaM inhibitor peptide attenuated a transient facilitation of I-pCa preceding inactivation observed at 500 Hz stimulation, whereas it had no effect on sustained I-pCa facilitations during trains of 50-200 Hz stimulation. These results suggest that the Ca2+/CaM-dependent I-pCa modulation requires a high intraterminal Ca2+ concentration, which can be attained at immature calyces during low frequency stimulation, but only during unusually high frequency stimulation at calyceal terminals in the posthearing period.

Paired-pulse facilitation (PPF) and depression (PPD) are forms of short-term plasticity that are generally thought to reflect changes in transmitter release probability. However, desensitization of postsynaptic AMPA receptors (AMPARs) significantly contributes to PPD at many glutamatergic synapses. To clarify the involvement of AMPAR desensitization in synaptic PPD, we compared PPD with AMPAR desensitization, induced by paired-pulse glutamate application in patches excised from postsynaptic cells at the calyx of Held synapse of developing rats. We found that AMPAR desensitization contributed significantly to PPD before the onset of hearing (P10-12), but that its contribution became negligible after hearing onset. During postnatal development (P7-21) the recovery of AMPARs from desensitization became faster. Concomitantly, glutamate sensitivity of AMPAR desensitization declined. Single-cell reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated a developmental decline of GluR1 expression that correlated with speeding of the recovery of AMPARs from desensitization. Transmitter release probability declined during the second postnatal week (P7-14). Manipulation of the extracellular Ca(2+)/Mg(2+) ratio, to match release probability at P7-8 and P13-15 synapses, revealed that the release probability is also an important factor determining the involvement of AMPAR desensitization in PPD. We conclude that the extent of involvement of AMPAR desensitization in short-term synaptic depression is determined by both pre- and postsynaptic mechanisms.

Molecular dependence of vesicular endocytosis was investigated with capacitance measurements at the calyx of Held terminal in brainstem slices. Intraterminal loading of botulinum toxin E revealed that the rapid capacitance transient implicated as "kiss-and-run" was unrelated to transmitter release. The release-related capacitance. change decayed with an endocytotic time constant of 10 to 25 seconds, depending on the magnitude of exocytosis. Presynaptic loading of the nonhydrolyzable guanosine, 5'-triphosphate (GTP) analog GTPgammaS or dynamin-1 proline-rich domain peptide abolished endocytosis. These compounds had no immediate effect on exocytosis, but caused a use-dependent rundown of exocytosis. Thus, the guanosine triphosphatase dynamin-1 is indispensable for vesicle endocytosis at this fast central nervous system (CNS) synapse.

Whether a quantal packet of transmitter saturates postsynaptic receptors is a fundamental question in central synaptic transmission. However, this question remains open with regard to saturation at mature synapses. The calyx of Held, a giant glutamatergic synapse in the auditory brainstem, becomes functionally mature during the fourth postnatal week in rats. During postnatal development, the mean amplitude of miniature (i.e., quantal) EPSCs (mEPSCs) becomes significantly larger. Experiments using the rapidly dissociating glutamate receptor antagonist kynurenate suggested that vesicular glutamate content increases with development. To test whether AMPA receptors are saturated by a packet of transmitter, we infused a high concentration of L-glutamate into mature calyceal terminals. This caused a marked increase in the mean amplitude of mEPSCs. We conclude that a single packet of transmitter glutamate does not saturate postsynaptic AMPA receptors even at the mature calyx of Held synapse with increased vesicular transmitter content.

The NMDA receptor (NMDAR) is a Ca2+-permeable cation channel that plays a critical role in neural network formation during brain development. Since it is blocked in a voltage-dependent manner by extracellular Mg2+, in order for the NMDA to be activated, the membrane must be strongly depolarized. Immature neurons in the developing neocortex can be depolarized by ligand-gated Cl- channels, such as the glycine receptor (GlyR) or GABA(A) receptor (GABA(A)R). We here assess the contribution of GlyRs to Ca2+ influx via NMDARs in neonatal mouse cortical neurons. The GlyR antagonist, strychnine, was more effective in suppressing postsynaptic Ca2+ influx than the GABA(A)R antagonist, picrotoxin, suggesting greater potentiation of NMDARs by GlyRs than by GABA(A)Rs. The GlyR, known to be endogenously activated at this stage, may play a critical role in neocortical development.