Jun Noguchi

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.

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.

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.

Evidence that multiple P2X purinoceptors are functionally expressed in rat supraoptic neurones

1 The expression, distribution and function of P2X purinoceptors in the supraoptic nucleus (SON) were investigated by reverse transcription‐polymerase chain reaction (RT‐PCR), in situ hybridization, and Ca2+‐imaging and whole‐cell patch‐clamp techniques, respectively. 2 RT‐PCR analysis of all seven known P2X receptor mRNAs in circular punches of the SON revealed that mRNAs for P2X2, P2X3, P2X4, P2X6 and P2X7 receptors were expressed in the SON, and mRNAs for P2X3, P2X4 and P2X7 were predominant. 3 In situ hybridization histochemistry for P2X3 and P2X4 receptor mRNAs showed that both mRNAs were expressed throughout the SON and in the paraventricular nucleus (PVN). 4 ATP caused an increase in [Ca2+]i in a dose‐dependent manner with an ED50 of 1.7 × 10−5m. The effects of ATP were mimicked by ATPγS and 2‐methylthio ATP (2MeSATP), but not by AMP, adenosine, UTP or UDP. αβ‐Methylene ATP (αβMeATP) and ADP caused a small increase in [Ca2+]i in a subset of SON neurones. 5 The P2X7 agonist 2′‐ & 3′‐O‐(4‐benzoylbenzoyl)‐ATP (BzATP) at 10−4m increased [Ca2+]i, but the potency of BzATP was lower than that of ATP. In contrast, BzATP caused a more prominent [Ca2+]i increase than ATP in non‐neuronal cells in the SON. 6 The effects of ATP were abolished by extracellular Ca2+ removal or by the P2 antagonist pyridoxal phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid (PPADS), and inhibited by extracellular Na+ replacement or another P2 antagonist, suramin, but were unaffected by the P2X7 antagonist oxidized ATP, and the inhibitor of Ca2+‐ATPase in intracellular Ca2+ stores cyclopiazonic acid. 7 Two patterns of desensitization were observed in the [Ca2+]i response to repeated applications of ATP: some neurones showed little or moderate desensitization, while others showed strong desensitization. 8 Whole‐cell patch‐clamp analysis showed that ATP induced cationic currents with marked inward rectification. The ATP‐induced currents exhibited two patterns of desensitization similar to those observed in the [Ca2+]i response. 9 The results suggest that multiple P2X receptors, including P2X3, are functionally expressed in SON neurones, and that activation of these receptors induces cationic currents and Ca2+ entry. Such ionic and Ca2+‐signalling mechanisms triggered by ATP may play an important role in the regulation of SON neurosecretory cells.

Transcranial optogenetic stimulation for functional mapping of the motor cortex.

We developed a method that uses Channelrhodopsin-2 (ChR2) for transcranial optogenetic stimulation. This method is based on scanning a light beam over the brain, thereby photostimulating ChR2-expressing neurons in intact mice. As a proof of principle, we applied this technique to the motor cortex of transgenic mice expressing ChR2 in cortical pyramidal cells. Photostimulation induced limb movements that were time-locked with millisecond precision and could be induced at frequencies up to 20 Hz. By scanning this light beam, we could map the distribution of neurons associated with limb movement. With this approach we could simultaneously define motor maps controlling two limbs and could reproducibly generate such cortical motor maps over periods of weeks. This method allows non-invasive mapping of brain circuitry in living animals and could help define the connection between behavior and brain circuitry.

GABA promotes the competitive selection of dendritic spines by controlling local Ca2+ signaling

Activity-dependent competition of synapses plays a key role in neural organization and is often promoted by GABA; however, its cellular bases are poorly understood. Excitatory synapses of cortical pyramidal neurons are formed on small protrusions known as dendritic spines, which exhibit structural plasticity. We used two-color uncaging of glutamate and GABA in rat hippocampal CA1 pyramidal neurons and found that spine shrinkage and elimination were markedly promoted by the activation of GABAA receptors shortly before action potentials. GABAergic inhibition suppressed bulk increases in cytosolic Ca2+ concentrations, whereas it preserved the Ca2+ nanodomains generated by NMDA-type receptors, both of which were necessary for spine shrinkage. Unlike spine enlargement, spine shrinkage spread to neighboring spines (<15 μm) and competed with their enlargement, and this process involved the actin-depolymerizing factor ADF/cofilin. Thus, GABAergic inhibition directly suppresses local dendritic Ca2+ transients and strongly promotes the competitive selection of dendritic spines.

In vivo two‐photon uncaging of glutamate revealing the structure–function relationships of dendritic spines in the neocortex of adult mice

Non‐technical summary  Neurons communicate with each other with synapses using chemical messengers. The major synapses in the cerebral cortex utilize glutamate as a messenger and are made on special submicron structures, called dendritic spines. Dendritic spines are diverse in their size and densely packed in the cortex. Therefore, an optical technique for application of glutamate to single spines (two‐photon (TP) uncaging) has been intensively used to clarify their functions in vitro. We have here extended 2P uncaging to living adult brain, and found that spine sizes display tight correlations with their functions, such as rapid glutamate sensing and an increase in cytosolic Ca2+ concentrations, even in vivo, as they were reported for in vitro preparations. Our data suggest that the structure and motility of dendritic spines play a key role in the adult brain function.

Learning rules and persistence of dendritic spines

Structural plasticity of dendritic spines underlies learning, memory and cognition in the cerebral cortex. We here summarize fifteen rules of spine structural plasticity, or ‘spine learning rules.’ Together, they suggest how the spontaneous generation, selection and strengthening (SGSS) of spines represents the physical basis for learning and memory. This SGSS mechanism is consistent with Hebb’s learning rule but suggests new relations between synaptic plasticity and memory. We describe the cellular and molecular bases of the spine learning rules, such as the persistence of spine structures and the fundamental role of actin, which polymerizes to form a ‘memory gel’ required for the selection and strengthening of spine synapses. We also discuss the possible link between transcriptional and translational regulation of structural plasticity. The SGSS mechanism and spine learning rules elucidate the integral nature of synaptic plasticity in neuronal network operations within the actual brain tissue.

PACAP increases the cytosolic Ca2+ concentration and stimulates somatodendritic vasopressin release in rat supraoptic neurons.

Pituitary adenylate cyclase activating polypeptide (PACAP)‐like immunoreactivity and its receptor mRNA have been reported in the supraoptic and the paraventricular nucleus (SON and PVN, respectively) and PACAP has been implicated in the regulation of magnocellular neurosecretory cell function. To examine the site and the mechanism of the action of PACAP in the neurosecretory cells, we measured AVP release from SON slice preparations and the cytosolic Ca2+ concentration ([Ca2+]i) from single dissociated SON neurons. PACAP at concentrations from 10−12 to 10−7 M increased [Ca2+]i in dissociated SON neurons in a dose‐dependent manner. The patterns of the PACAP‐induced [Ca2+]i increase were either sustained increase or cytosolic Ca2+ oscillations. PACAP (10−7 M) increased [Ca2+]i in 27 of 27 neurons and glutamate (10−4 M) increased [Ca2+]i in 19 of 19 SON neurons examined, whereas angiotensin II (10−7 M) increased [Ca2+]i in only 15 of 60 SON neurons examined. PACAP at lower concentrations (10−10 to 10−8 M) increased [Ca2+]i in 70–80% of neurons examined. Although the onset and recovery of the PACAP‐induced [Ca2+]i increase were slower than those observed with glutamate, the spatial distribution of the [Ca2+]i increases in response to the two ligands were similar: [Ca2+]i increase at the proximal dendrites was larger and faster and that at the center of the soma was smaller and slower. The PACAP‐induced [Ca2+]i responseswere abolished by extracellular Ca2+ removal, the l‐type Ca2+‐channel blocker, nicardipine, or by replacement of extracellular Na+ with N‐methyl d‐glucamine, and were partially inhibited by the Na+‐channel blocker, tetrodotoxin. The N‐type Ca2+‐channel blocker, ω‐conotoxin GVIA did not significantly inhibit the PACAP‐induced [Ca2+]i responses. Furthermore, PACAP (10−7 M) as well as glutamate (10−4 M) increased AVP release from SON slice preparations, and extracellular Ca2+ removal or nicardipine inhibited the AVP release in response to PACAP.

SNARE Conformational Changes that Prepare Vesicles for Exocytosis

When cells release hormones and neurotransmitters through exocytosis, cytosolic Ca(2+) triggers the fusion of secretory vesicles with the plasma membrane. It is well known that this fusion requires assembly of a SNARE protein complex. However, the timing of SNARE assembly relative to vesicle fusion--essential for understanding exocytosis--has not been demonstrated. To investigate this timing, we constructed a probe that detects the assembly of two plasma membrane SNAREs, SNAP25 and syntaxin-1A, through fluorescence resonance energy transfer (FRET). With two-photon imaging, we simultaneously measured FRET signals and insulin exocytosis in beta cells from the pancreatic islet of Langerhans. In some regions of the cell, we found that the SNARE complex was preassembled, which enabled rapid exocytosis. In other regions, SNARE assembly followed Ca(2+) influx, and exocytosis was slower. Thus, SNARE proteins exist in multiple stable preparatory configurations, from which Ca(2+) may trigger exocytosis through distinct mechanisms and with distinct kinetics.