Workshop
Frontiers in Cellular Neuroimaging
June 17-22, 2005
RIKEN Brain Science Institute
Wako-shi, Saitama, Japan
http://www.brain.riken.go.jp/events/neuroimage/
Organizing committee
Alexey Semyanov
Hajime Hirase
Thomas Knöpfel
PROGRAM
Friday, June 17, 2005
10:00 - 17:00
Registration in Ikenohata building and check in at RIKEN International House
12:00-13:30
Lunch (RIKEN canteen)
15:30 – 16:00
Opening and welcome address
Ikenohata Building
Shun-ichi Amari, Director of RIKEN Brain Science Institute
16:00 - 17:00
Plenary lecture : Alan Fine (National Institute for Medical Research, UK and
Dalhousie University Faculty of Medicine, Canada)
Imaging the function and plasticity of individual synapses in the hippocampus
18:00 – 20:00
Dinner (RIKEN cafeteria)
2
Saturday, June 18, 2005
Session 1 (Chair: Kohtaro Kamino)
Okochi Hall
10:00 - 11:00
Larry Cohen (Yale University School of Medicine, USA)
1.1 The input from the nose to the olfactory bulb; and the resulting oscillations
11:00 - 12:00
Kenichi Ohki (Harvard Medical School, USA)
1.2 Two-photon calcium imaging reveals precise functional micro-architecture in
visual cortex
Lunch (BSI coffee shop)
12:00 - 14:00
14:00 - 15:00
Gero Miesenboek (Yale University School of Medicine, USA)
1.3 Optical control of neuronal circuits and behavior
15:00 - 16:00
Selected talks by participants (20 min each)
Nikolai Otmakhov (Brandeis University, Waltham, USA)
1. Persistent accumulation of CaM Kinase II in dendritic spines after induction
of LTP
ML Renner (University of Bordeaux-2, Bordeaux, France)
2. Probing membrane dynamics in neuronal synapses
16:00 - 16:30
16:30 - 17:30
Ryota Homma (RIKEN Brain Science Institute, Japan)
3. Voltage-sensitive dye imaging in vivo revealed a novel functional structure in
monkey IT cortex
Coffee break
Atsuo Fukuda (Hamamatsu University School of Medicine, Japan)
1.4 Monitoring the dynamics of neural functions modulated by intracellular Cl-
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Sunday, June 19, 2005
Session 2 (Chair: Hajime Hirase)
Okochi Hall
10:00 - 11:00
Atsushi Miyawaki (RIKEN BSI, Japan)
2.1 Visualization of the spatial and temporal dynamics of intracellular signaling
11:00 - 12:00
Yury Kovalchuk (LMU, Munich, Germany)
2.2 High-speed two-photon imaging of neuronal activity
12:00 - 14:00
Lunch (BSI coffee shop)
14:00 - 15:00
Rosa Cossart (INMED, Marseille, France)
2.3 Fast two-photon calcium imaging of network activity: “watching” thousands of
neurons “at work”
15:00 - 16:00
16:00 - 16:30
Poster session
Coffee break
16:30 - 17:30
Ann-Shyn Chiang (National Tsing Hua University, Taiwan)
2.4 Mapping memory circuits in the drosophila brain
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Monday, June 20, 2005
Session 3 (Chair: Alexey Semyanov)
Okochi Hall
10:00 - 11:00
Dmitri Rusakov (Institute of Neurology, London, UK)
3.1 Activity-dependent control of rapid presynaptic Ca2+ signalling at individual
central synapses
11:00 - 12:00
Yuji Ikegaya (The University of Tokyo, Japan)
3.2 Imaging the extracellular dynamics of Zn2+
Lunch (RIKEN cafeteria)
12:00 - 14:00
14:00 - 15:00
Thomas Knöpfel (RIKEN BSI, Japan)
3.3 Voltage and ion-imaging: Genetically encoded probes versus organic chemistry
15:00 - 16:00
Selected talks by participants (20 min each)
Ricardo Scott (Institute of Neurology, UCL, London, UK)
1. Presynaptic Ca2+ signalling at individual mossy fibre synapses
Ryosuke Enoki (National Institute for Medical Research, London, UK)
2. The origin of synaptically-evoked Ca2+ transients in hippocampal dendritic spines
16:00 - 16:30
16:30 - 17:30
P.M. Balaban (Institute of Higher Nervous Activity and Neurophysiology, Moscow,
Russia)
3. Correlation between optically recorded Ca2+ influx in one presynaptic terminal and
amplitude of PSP in identified synapse in vivo
Coffee break
Round table discussion (Chair: Thomas Knöpfel)
Voltage and ion-imaging: Genetically encoded probes versus organic chemistry
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Tuesday, June 21, 2005
Session 4 (Chair: Thomas Knöpfel)
Okochi Hall
10:00 - 11:00
Daniel Choquet (University of Bordeaux 2, Cellular Physiology of Synapse, France)
4.1 Single molecule imaging of glutamate receptor trafficking in and out synapses
11:00 - 12:00
Leonard Khiroug (Helsinki University, Finland)
4.2 Applications of local photolysis of intra- and extracellular caged compounds
12:00 - 14:00
Lunch (RIKEN cafeteria)
14:00 - 15:00
Tadaharu Tsumoto (RIKEN BSI, Japan)
4.3 Activity-regulated trafficking of BDNF in axons and dendrites of cortical neurons
15:00 - 16:00
Katsushige Sato (Tokyo Medical and Dental University, Japan)
4.4 Functional visualization of emerging brainstem neural
voltage-sensitive dye imaging
18:00 – 20:00
Sayonara dinner at RIKEN Campus
Tickets should be purchased in advance
6
circuits
using
Wednesday, June 22, 2005
9:00
Check out in I-house and departure
7
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LECTURE ABSTRACTS
Plenary lecture: Imaging the function and plasticity of individual synapses in the hippocampus
Alan Fine
National Institute for Medical Research, UK and Dalhousie University Faculty of Medicine, Canada
The application of fluorescent Ca2+ indicators and laser-scanning microscopy to hippocampal slice
preparations has made it possible to monitor transmission at individual visualized synapses. This
approach has enabled us to uncover significant and functional Ca2+-induced Ca2+ release from internal
stores on both sides of the synapse, as well as functional synaptically-evoked depletion of Ca2+ from the
synaptic cleft. Here, I will concentrate on recent work in which we have used synaptically-evoked Ca2+
transients in dendritic spines to carry out optical quantal analysis of transmission at individual synapses
before and after the induction of long-term synaptic plasticity. We find that individual synapses can
sustain incremental (as opposed to binary) and bi-directional plasticity. Both LTP and LTD are expressed
in large part through graded changes in the probability of transmitter release; thus changes in glutamate
receptors appear not to be the dominant mechanisms for expression of plasticity. Expression of LTP or
LTD under physiological conditions is not associated with formation or loss of dendritic spines, nor with
detectable changes in spine morphology, up to at least 4h after induction of plasticity; thus such structural
changes as have been previously reported appear not to be essential components of long-term synaptic
plasticity.
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1.1 The input from the nose to the olfactory bulb; and the resulting oscillations
Larry Cohen
Yale University School of Medicine, USA
Olfactory receptor neurons project axons to specific glomeruli of the olfactory bulb so that all the
sensory input collected by a single type of olfactory receptor protein converges onto only two glomeruli
located on opposite sides of the bulb. To visualize odorant representations by receptor neuron input to the
mouse olfactory bulb, we loaded olfactory receptor neurons with a calcium-sensitive dye and imaged
odorant-evoked fluorescence increases from their axon terminals in vivo. The odorants we tested
activated many glomeruli at reasonably low odorant concentrations. Different odorants had different
maps of activated glomeruli. The maps were concentration dependent; increasing odorant concentrations
activated an increasing number of glomeruli. Thus the maps of input to the olfactory bulb are a confound
of odor quality and concentration. Five glomeruli were identifiable across animals; all five showed
complex response specificities to a set of 16 odorants.
One of the functions of the olfactory bulb may be to sparsen this rather widespread activation of
glomeruli. We present evidence that centre-surround inhibition shapes the odour-evoked input to the
bulb in vivo. Strongly activated glomeruli suppress calcium influx into axon terminals of olfactory
receptor neurons terminating in the surrounding glomeruli. This lateral inhibition has the effect of
increasing contrast of the sensory input map.
Odorants elicit oscillations in the olfactory bulb. We measured the spatio-temporal aspects of
these oscillations in the in vivo turtle bulb using a voltage-sensitive dye, RH414. We distinguished three
different oscillations. These oscillations occurred over broad regions of the bulb. The initial frequency of
the three oscillations were 14.1 Hz, 13.0 Hz, and 6.6 Hz respectively. When the rostral and caudal
oscillations occurred together their frequencies differed by a factor of 1.99 + 0.01.
We found that dramatic alterations in the oscillatory pattern occur on repeated odorant presentations.
The changes to two of the oscillations, rostral and caudal, are odorant independent; in contrast, the
change observed in the middle oscillation depends on whether the odorant in the two presentations is the
same or different. Thus, oscillations may have more than one role during odorant processing; the middle
oscillation could be used as a detector of stimulus novelty while the rostral and caudal oscillations may
be part of the mechanism for odor accomodation.
Supported by NIH grant DC05259.
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1.2 Two-photon calcium imaging reveals precise functional micro-architecture in visual cortex
Kenichi Ohki
Harvard Medical School, USA
Neurons in the cerebral cortex are organized into anatomical columns, with ensembles of cells
arranged from the surface to the white matter. Within a column, neurons often share functional properties,
such as selectivity for stimulus orientation; columns with distinct properties, such as different preferred
orientations, tile the cortical surface in orderly patterns. This functional architecture was discovered with
the relatively sparse sampling of microelectrode recordings. Optical imaging of membrane voltage or
metabolic activity elucidated the overall geometry of functional maps, but is averaged over many cells
(resolution >100 micron). Consequently, the purity of functional domains and the precision of the
borders between them could not be resolved. Here, we labelled thousands of neurons of the visual cortex
with a calcium-sensitive indicator in vivo. We then imaged the activity of neuronal populations at
single-cell resolution with two-photon microscopy up to a depth of 400 micron. In rat primary visual
cortex, neurons had robust orientation selectivity but there was no discernible local structure;
neighbouring neurons often responded to different orientations. In area 18 of cat visual cortex, functional
maps were organized at a fine scale. Neurons with opposite preferences for stimulus direction were
segregated with extraordinary spatial precision in three dimensions, with columnar borders one to two
cells wide. These results indicate that cortical maps can be built with single-cell precision.
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1.3 Optical control of neuronal circuits and behavior
Gero Miesenbock
Department of Cell Biology, Yale University School of Medicine, USA
Optically gated ion channels were expressed in circumscribed groups of neurons in the Drosophila
CNS, so that broad illumination of flies evoked action potentials only in genetically designated target
cells. Flies harboring the “phototriggers” in different sets of neurons responded to laser light with
behaviors specific to the sites of phototrigger expression. Photostimulation of neurons in the giant fiber
system elicited the characteristic escape behaviors of jumping, wing beating, and flight; photostimulation
of dopaminergic neurons caused changes in locomotor activity and locomotor patterns. These responses
reflected the direct optical activation of central neuronal targets rather than confounding visual input, as
they persisted unabated in carriers of a mutation that eliminates phototransduction. Encodable
phototriggers provide non-invasive control interfaces for studying the connectivity and dynamics of
neural circuits, for assigning behavioral content to neurons and their activity patterns, and, potentially,
for restoring information corrupted by injury or disease.
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1.4 Monitoring the dynamics of neural functions modulated by intracellular ClAtsuo Fukuda
Hamamatsu University School of Medicine, Japan
One of recent topics in neuroscience is that GABA necessarily acts excitatory (Cl - efflux) in
immature brain, in contrast to inhibitory (Cl- influx) in normal adult brain. Such excitatory GABA
actions may be involved in neural circuitry development. On the other hand, a conversion of GABA
response from inhibitory to excitatory could be induced by certain pathological condition even in adult.
Such developmental and pathological GABAergic inhibition-excitation switches are induced by changes
in Cl- gradient generated by cation-Cl- cotransporters (e.g., KCC2 and NKCC1). Since changes in
intracellular Cl- do not always mean such a switch, e.g., shunting inhibition by depolarizing GABA, we
should combine a variety of techniques to assess functional dynamics of intracellular Cl-.
By using Cl- imaging in brain slices, a technique which enables visualization of dynamic [Cl -]i
changes in individual neurons, we can study dynamic and spatial alterations in Cl - homeostasis.
Gramicidin-perforated patch-clamp recordings followed by single-cell RT-PCR enable us to estimate
[Cl-]i directly and to identify specific molecules responsible for the changes in Cl - homeostasis and
GABA responses in question. We also studied changes in [Ca2+]i and membrane potential responses to
GABA as well as changes in resting [Cl-]i, to evaluate local neural circuitry alterations induced by
changes in [Cl-]i. In addition, visualization of particular population of neurons by in utero
electroporation of plasmids encoding fluorescent protein and/or Cl- transporters help us investigate nature
of the cells modulated by intracellular Cl-. Thus, we are investigating dynamics of neural functions
modulated by intracellular Cl- by means of optical imaging (Cl-, Ca2+, voltage and GFP) and
electrophysiological methods. As examples, I introduce some of our applications of imaging techniques
on evaluation of dynamics of neural functions modulated by intracellular Cl- as below.
In individual neurons in brain slices in which Cl--sensitive fluorescent dye MEQ was injected from
patch electrode, dynamic alterations in the inhibitory synaptic function and [Cl-]i was monitored by
simultaneous optical and electrophysiological recordings of [Cl-]i and membrane potential. An acute Cloverload induced by high-frequency stimulation and resultant convergence of GABAergic action from
inhibition to excitation was revealed. Optical imaging of membrane potential using voltage-sensitive
dye was also used to assess dynamic changes in network function driven by depolarizing GABA actions.
We found that axotomy induces spontaneous [Ca2+]i oscillation, supersensitivity to glutamate-evoked
[Ca2+]i rises and GABA-evoked [Ca2+]i elevations in motoneurons. The increases in [Cl-]i induced by
KCC2 downregulation was assessed by Cl- imaging and gramicidin patch to prove that elevation of [Cl-]i
turned GABAergic inhibition to excitation and induced spontaneous [Ca2+]i oscillations. Furthermore,
to prove that high [Cl-]i and resultant excitatory GABA actions contribute to cell migration, we applied in
utero electroporation for over-expression of KCC2 to reduce [Cl-]i.
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2.1 Visualization of the spatial and temporal dynamics of intracellular signaling
Atsushi Miyawaki
RIKEN Brain Science Institute, Japan
“Why bio-imaging, i.e. real time fluorescence imaging?” Currently, this is a topic of great interest in
the bioscience community. Many molecules involved in signal transduction have been identified, and the
hierarchy among those molecules has also been elucidated. It is not uncommon to see a signal
transduction diagram in which arrows are used to link molecules to show enzyme reactions and
intermolecular interactions. To obtain a further understanding of a signal transduction system, however,
the diagram must contain the three axes in space as well as a fourth dimension, time, because all events
are controlled ingeniously in space and time. Since the isolation of green fluorescent protein (GFP) from
the bioluminescent jellyfish in 1992 and later with its relatives, researchers have been awaiting the
development of a tool which enables the direct visualization of biological functions. This has been
increasingly enhanced by the marriage of GFP with fluorescence resonance energy transfer (FRET), and
is further expanded upon by the need for “post-genomic analyses.” It is not my intent to discourage the
trend seeking the visualization of biological function. I would like to propose that it is time to evaluate
the true asset of “bio-imaging” for its potential and limitations in order to utilize and truly benefit from
this novel technique.
Our recent study provides an important guide for the development and improvement of indicators
using GFP-based FRET. Yellow cameleon (YC) is a Ca2+ indicator, which has cyan- and yellow-emitting
fluorescent proteins (CFP and YFP). We optimized the relative orientation of the two chromophores by
fusing YFP at different angles. We generated circularly permuted YFPs (cpYFPs) that showed efficient
maturation and acid stability. One of the cpYFPs incorporated in YC absorbes a great amount of excited
energy from CFP in its Ca2+-saturated form, thereby increasing the Ca2+-dependent change in the ratio of
YFP/CFP by nearly 600%.
We may move towards a more comprehensive understanding of signal transduction systems. I will
present the direct evidence that astrocytes affect neuronal synaptogenesis by the process of adhesion.
Local contact with astrocytes via integrin receptors elicited protein kinase C (PKC) activation in
individual dissociated neurons cultured in astrocyte-conditioned medium. This activation, initially focal,
soon spread throughout the entire neuron. We then demonstrated pharmacologically that the arachidonic
acid cascade, triggered by the integrin reception, is responsible for the global activation of PKC. Local
astrocytic contact also facilitated excitatory synaptogenesis throughout the neuron, a process which could
be blocked by inhibitors of both integrins and PKC. Thus, propagation of PKC signaling represents an
underlying mechanism for global neuronal maturation following local astrocyte adhesion.
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2.2 High-speed two-photon imaging of neuronal activity
Yury Kovalchuk
LMU, Munich, Germany
Conventionally two-photon laser-scanning microscopy (TPM ) is performed by using 100
femtosecond-wide laser pulses directed through scanning galvano-metric mirrors. Such systems require
typically 1 s for a full frame scan , whereas many processes in living tissues occur at much faster time
scale. High-speed scanning acousto-optic deflectors (AODs), although used successfully for
single-photon imaging, are considered to be ill-suited for TPM, because the spectral bandwidth of the
femtosecond laser pulses is considerably dispersed by the acoustic wave inside the crystal.
Using a high-power Ti:sapphire laser with a picosecond pulse-length and the AOD-based scanning
system we succeeded to perform high-speed TPM in living tissue. Up to 20 mW excitation light power
was achievable under the 60x objective and provided high-quality two-photon images of individual
neurones in brain slices including their finest dendrites and dendritic spines. In our system full frames
were acquired at video rate, but acquisition speed of up to 480 sub-frames/s was achievable, resulting in
a pixel dwell time as short as 100 ns.
A comparable dwell time was also achieved in another scanning system which utilises resonant
scanning mirror. Both systems provide sufficient spatial and temporal resolution to study Ca 2+ signals
caused by back-propagating action potentials and synaptic events, as well as to study the activity of many
individual cells within a neuronal network in vitro and in vivo.
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2.3. Fast two-photon calcium imaging of network activity: “watching” thousands of neurons “at
work
Rosa Cossart
Institut de Neurobiologie de la Mediterranee, Marseilles, France
Network activities usually involve a large number of cells that are extremely heterogeneous in terms
of their morpho- physiological properties and neurochemical content; therefore, it is essential to have a
dynamical picture of the behaviour of the entire network and to characterize simultaneously the activity
of its individual components at the single-cell level. The considerable progress made in imaging
techniques allows us for the first time to have simultaneously a dynamic and global characterization of
network activity and to determine the single-cell properties of the unitary microcircuits involved in this
activity (Cossart et al. Nature 2003, Ikegaya et al. Science 2004). We use calcium transients to
reconstruct action-potential activity since there is a strict correlation between the firing and the calcium
entry inside a given neuron. The method we developed is based on two-photon calcium imaging of large
neuronal populations; it achieves singe-cell resolution and is associated to automated signal processing
techniques that enable to perform online analysis of activity in over 1500 neurons simultaneously
(Cossart et al. Nature 2003, Ikegaya et al., Science 2004). However, the major limitation of such method
based on conventional scanners is time resolution. Indeed, the typical frame rate being around 1 sec for a
full frame (512X512), it is impossible to detect reliably the occurrence of an action potential or to
discriminate between two consecutive events. We are therefore using a pioneering confocal system based
on a multibeam scanning of the preparation that achieves millisecond resolution.
We study the structure of network activity in the visual cortex immature hippocampus, using a
double experimental strategy: an integrated approach using ultrafast two-photon microscopy to image
calcium transients with millisecond resolution to reconstruct the dynamics of the entire network activity
with single-cell resolution, combined with a unitary approach using whole-cell multiple patch clamp
recordings to characterize the physiological properties of the individual key elements implicated in the
network activity.
I will present data regarding the spontaneous activity that takes place in the mouse visual cortex and
show that in the absence of any stimulation, groups of cortical neurons simultaneously enter membrane
potential UP states. These ‘network UP states’ involve small, but highly significant numbers of cells
(2-3%). Coactive ensembles of neurons are often organized into clusters, layers, or columns. The
stereotyped spatiotemporal dynamics of this activity are highly reminiscent of those theoretically
predicted for feedback and recurrent neural networks. Furthermore, we investigated the synaptic and
intrinsic mechanisms of network UP states and show that the GABAergic network may be responsible
for generating the network synchronizations we observe.
We propose that the coordinated activity of specific interneuron subclasses provides a circuit
mechanism for triggering the stereotyped occurrence of network UP states.
In a second part of my talk I will present essential information to better understand the
morpho-functional structure of developing hippocampal network.
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2.4. Mapping Memory Circuits in the Drosophila Brain
Ann-Shyn Chiang
Brain Research Center, National Tsing Hua University, Taiwan
Thousands of fly genes operate in the brain circuits, each at a different time and space, controlling
complex behavior. While Gal4/UAS reporter system and molecular tagging techniques have led a useful
model of how gene products work, the expression patterns of single gene and lacking enough resolution
to visualize brain circuits are limited in giving us a comprehensive picture of the brain’s higher functions
such as learning and memory. We reason that if all connections among neurons could be mapped,
together with genes expressed in these neurons at different times could be visualized in the whole brain,
the resulting spatial and temporal circuit diagrams should reveal mechanisms underlying the brain's
operation. The challenge is to integrate this multidimensional information in a way that will help
biologists to find meaningful relationships in the data and to formulate creative hypotheses that can be
tested by further observation and experiment. Modern studies of the genetic control of memory have
increased the need for an accurate and comprehensive storage and display of gene expression data. Here,
I summarize our recent progress in (1) development of new imaging methods allowing visualization of
gene expression patterns and individual brain circuits in the whole brain at high resolution, (2) analysis
the direction of information flow indicated by circuit polarity, (3) the generation of a common digital
framework for cataloging 3D images of individual neurons expressed with specific genes, (4) interactive
internet utilization of an alpha version of 3D Circuitry Database, (5) molecular and behavior verification
of memory circuits.
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3.1. Activity-dependent control of rapid presynaptic Ca2+ signalling at individual central synapses
Dmitri A. Rusakov
Institute of Neurology, University College London, Queen Square WC1N 3BG, London, UK
Rapid, activity-driven modulation of Ca2+-dependent synaptic release by presynaptic receptors
contributes critically to the fundamental mechanisms of information processing in the brain. To probe
these mechanisms at a single-synapse level, we combined confocal / two-photon microscopy with
single-cell electrophysiology in acute brain slices. We monitored and analysed fast, action potential
evoked Ca2+ transients in several types of individual presynaptic terminals that represent major synaptic
circuitries in the hippocampus and cerebellum. We identified sub-cellular mechanisms by which synaptic
release is regulated through activation of (a) presynaptic GABAA receptors at glutamatergic synapses
formed by hippocampal mossy fibres and (b) presynaptic glutamate receptors (AMPA and group III
metabotropic types) in GABAergic terminals of hippocampal and cerebellar interneurons. The ability to
probe individual synapses reveals important organisation principles that contribute to the differentiation
of synaptic release control within the apparently homogenous synaptic populations.
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3.2. Imaging the extracellular dynamics of Zn2+
Yuji Ikegaya
The University of Tokyo, Japan
Zn2+, one of the most abundant divalent metal ions in the central nervous system (CNS), is known to
modulate postsynaptic neurotransmitter receptor activity. For instance, it inhibits NMDA receptors and
γ-aminobutyric acid receptors, and potentiates AMPA receptors. Zn2+ is also able to permeate
ligand-gated channels, e.g., NMDA receptor channels, Ca2+-permeable AMPA/kainate receptor channels
and voltage-dependent Ca2+ channels, and may influence various intracellular signaling pathways. Zn2+
is mainly stored in the synaptic vesicles of excitatory synapses, in particular the synaptic terminals of
hippocampal mossy fibers (MFs), and is co-released with neurotransmitters in response to synaptic
activity, but its physiological role in synaptic transmission is poorly understood. By using the newly
developed high-sensitivity Zn2+ indicator ZnAF-2, the spatiotemporal dynamics of Zn2+ was monitored in
rat hippocampal slices. When high-frequency stimulation was delivered to the MFs, the concentration
of extracellular Zn2+ was immediately elevated in the stratum lucidum, followed by a mild increase in the
stratum radiatum adjacent to the stratum lucidum, but not in the distal area of stratum radiatum. The
Zn2+ increase was insensitive to a non-NMDA receptor antagonist but was efficiently attenuated by
tetrodotoxin or Ca2+-free medium, which suggests that Zn2+ is released by MF synaptic terminals in an
activity-dependent manner and thereafter diffuses extracellularly into the neighboring stratum radiatum.
Electrophysiological analyses revealed that NMDA-receptor-mediated synaptic responses in CA3
proximal stratum radiatum were inhibited in the immediate aftermath of MF activation and that this
inhibition was no longer observed in the presence of a Zn2+-chelating agent. Thus, Zn2+ serves as a
spatiotemporal mediator in imprinting the history of MF activity in contiguous hippocampal networks.
We predict herein a novel form of metaplasticity, i.e., an experience-dependent ‘non-Hebbian’
modulation of synaptic plasticity.
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3.3. Voltage and ion-imaging: Genetically encoded probes versus organic chemistry
Thomas Knöpfel
RIKEN Brain Science Institute, Japan
During the last few decades, powerful functional optical imaging techniques have been developed
that cover a spatial scale from subcellular to the system level. Best imaging results are usually obtained
with fluorescence-based measurements that require (i) the use of an organic chemistry-based fluorescent
dye, or (ii) the use of a fluorescent molecule that is naturally present in brain tissue or (iii) that the tissue
expresses a genetically encoded fluorescent protein.
Our laboratory has used organic-chemistry based neuroimaging over many years and more recently
started to pioneer the use of activity dependent autofluorescence and genetically encoded probes for
membrane voltage and intracellular calcium concentration.
At the level of single cells, we have used the sodium-sensitive dye SBFI and the calcium sensitive
fluorescent dye Oregon green BAPTA-1 to investigate intrinsic properties and mechanisms of integration
of synaptic inputs in cerebellar Purkinje cells and olfactory mitral cells.
At the level of neuronal circuits, we have used voltage sensitive dyes (di-4-ANEPPS), activity
dependent autofluorescence signals and a genetically-encoded calcium sensor protein to image the
synaptic information flow in the cerebellar mossy fiber-granule cell-Purkinje cell pathway.
The above research results will be used to exemplify issues related to the methodology and
application of optical imaging of CNS networks.
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4.1 Single molecule imaging of glutamate receptor trafficking in and out synapses
Daniel Choquet (1), L. Groc (1), M. Heine (1), M. Renner (1), C. Bats (1), L. Cognet (2), B. Lounis (2)
1) CNRS UMR 5091 University of Bordeaux-2, Bordeaux, France
2) CNRS UMR 5798 University of Bordeaux-1, Talence, France.
A central process in learning and memory formation is the modification of synaptic strength by
changing the amount of glutamate receptors present at synapses. We will show that receptors exchange
between synaptic and extrasynaptic spaces by lateral diffusion and that this might play an important role
for the regulation of synaptic transmission. We applied single molecule tracking to follow glutamate
receptor and lipid dynamics in living neurons during activity driven changes in synaptic transmission. We
directly imaged AMPA and NMDA type glutamate receptor movements inside, at the periphery and
outside synapses of live cultured hippocampal neurons using single-molecule fluorescence microscopy.
In conditions of glutamate induced synaptic depression, receptor mobility increased inside synapses.
Conversely, during the processes which lead to increased AMPARs numbers at synapses such as high
frequency electrical stimulation, receptors are initially mobile in synapses and are then stabilized.
Altogether, our results establish than synapses are specialized membrane microdomains whose
components display a much higher dynamic than previously thought. This dynamic may explain the
rapid changes in receptor composition observed during the processes of synaptic plasticity.
Borgdorff, A. and Choquet, D. (2002). Nature, 417, 649–653; Groc, L.,et al. (2004). Nat Neurosci 7,
695-696 ; Triller, A., and Choquet, D. (2005). Trends Neurosci 28, 133-139.
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4.2. Applications of local photolysis of intra- and extracellular caged compounds
Leonard Khiroug
Helsinki University, Finland
In neurophysiological studies, including neuroimaging and electrophysiological experiments,
signaling mechanisms are often assayed by quickly changing the concentration of a ligand (or an ion) in
the vicinity of the receptor in question. Failure to deliver the ligand with a sufficiently rapid onset often
results in serious misinterpretation of the cellular responses, e.g. due to receptor pre-desensitization.
While cultured cells and, in particular, excised membrane patches have been successfully studied with
rapid solution exchange systems, fast modification of ligand or ion concentration may be extremely
difficult in the more physiological preparations such as brain slices or intact brains. This obstacle can be
overcome by applying a “caged” form of the ligand or ion to the receptor vicinity. The caged compound
will remain biologically inert until photo-activated with a brief pulse of the UV light. The photolytic
“uncaging” achieved with a focused UV laser beam results in a rapid (sub-millisecond range) and local
(micrometer range) increase in the concentration of a desired ligand or ion. When caged agonists are
applied extracellularly, their uncaging can produce cellular responses which closely mimic the effects of
synaptically released neurotransmitters. Moreover, intracellular delivery of caged Ca 2+ or other caged
second messengers allows manipulation of signal transduction mechanisms in a spatially and temporally
controlled manner. Applications of local uncaging technique for functional receptor mapping, synaptic
plasticity regulation, subcellular ion gradient measurements, and studies of intracellular Ca2+ signaling
will be discussed.
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4.3. Activity-regulated trafficking of BDNF in axons and dendrites of cortical neurons
Tadaharu Tsumoto
RIKEN Brain Science Institute, Japan
Brain-derived neurotrophic factor (BDNF) is suggested to play a role in neuronal development and
synaptic plasticity. Important steps of processes for BDNF to exert such a role are assumed to be its
trafficking in neurites to release sites, activity-dependent release and binding with receptors at
postsynaptic neurons or presynaptic terminals. Elucidation of mechanisms underlying such dynamic
movements of BDNF is crucial for understanding the function of BDNF.
To address this issue, we developed time-lapse imaging techniques to demonstrate intra- and
inter-cellular trafficking of BDNF tagged with green fluorescent protein (GFP) or other fluorescent
proteins in living cortical neurons with the method of direct intranuclear injection of plasmid cDNAs.
With this method we found that BDNF moves in presynaptic axons in the anterograde direction and
transfers to postsynaptic neurons in an activity-dependent manner (Kohara et al., Science 291, 2419,
2001; J Neurosci 23, 6123, 2003).
Then, we address a question of whether the intracellular trafficking of BDNF is regulated by
neuronal activity. With the time-lapse imaging technique we quantitatively analyzed movements of
BDNF in axons and dendrites of living cortical neurons. We found that BDNF-containing vesicles
moved smoothly in axons at the mean velocity of 0.73 ± 0.26 μm/sec while those in dendrites did not
move smoothly, i.e., often stopped or moved back and forth in most cases. When glutamate (100 μM)
was applied, the movement of vesicles in neurites was stopped and the intensity of their fluorescence
was reduced in most cases, suggesting an activity-induced stop-and-release of BDNF. An application of
antagonists for glutamate receptors or a replacement of the external medium by the Ca2+-free solution
blocked such an action of glutamate. A question of how activity and a subsequent influx of Ca2+ regulate
BDNF trafficking will be discussed in this talk.
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4.4 Functional visualization of emerging brainstem neural circuits using voltage-sensitive dye
imaging
Katsushige Sato (1), Yoko Momose-Sato (1), Kohtaro Kamino (1) and Joel C. Glover (2),
1) Tokyo Medical and Dental University Graduate School and Faculty of Medicine, Tokyo, Japan
2) University of Oslo School of Medicine, Oslo, Norway
The ontogenetic approach to physiological events is a useful strategy for understanding the
functional organization/architecture of vertebrate nervous systems.
However, conventional
electrophysiological techniques are difficult or impossible to employ with early embryonic neurons. In
addition, the electrophysiological analysis of firing patterns of individual neurons does not provide
information about the extent or spatio-temporal patterns of neuronal activity. Optical techniques using
voltage-sensitive dyes have made it possible to monitor electrical events in embryonic neurons that are
inaccessible to microelectrodes, and have proven to be a useful tool for analyzing the developmental
expression of neural functions and their spatio-temporal dynamics.
Using the optical recording technique, we have investigated the developmental organization of neural
circuits related to the cranial and spinal nerves in the chick and rat embryos. The aim of this study is to
find the fundamental principles of neural circuit formation in the central nervous system. As easily
accessible models, we focused on the brainstem neural circuits related to the trigeminal nerve (N. V:
general somatic nerve), vestibulo-cochlear nerve (N. VIII: special somatic nerve) and vagus nerve (N. X:
general and special visceral nerve). From developmental pursuits of the brainstem network formation,
we found that (1) in the 1st-ordered nucleus, postsynaptic responses are observed from early stages of
development, before differentiation of pre- and postsynaptic neurons has been completed and the
morphological structure of conventional synapses has appeared; (2) neural excitability and synaptic
function in a higher-ordered nucleus have already been generated by the time synaptic function in the
first-ordered nucleus is initially expressed; and (3) connections between the first- and higher-ordered
nuclei are established in a manner similar to the adult pattern from the beginning of nuclear organization.
These results demonstrate that the optical recording technique provides a rapid means of assessing the
functional connectivity of neural circuits during embryonic development.
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SHORT TALK ABSTRACTS
June 18
1. Persistent accumulation of CaM Kinase II in dendritic spines after induction of LTP
Nikolai Otmakhov, Jung-Hwa Tao-Cheng, Stephen Carpenter, Brent Asrican, Ayse Dosemeci, Thomas S.
Reese and John Lisman
Brandeis University, Waltham, USA
CaMKII is a leading candidate for a synaptic memory molecule because it is persistently activated
after LTP induction and because mutations that block this persistent activity prevent LTP and learning.
Previous work has shown that synaptic stimulation causes a rapidly reversible translocation of CaMKII
to the synaptic region. We have now measured GFP-CaMKIIalpha translocation into synaptic spines
during NMDAR-dependent chemical LTP (cLTP) and find that under these conditions translocation is
persistent. Using red fluorescent protein as a cell morphology marker, we found that there are two
components of the persistent accumulation. cLTP produces a persistent increase in spine volume and
some of the increase in GFP-CaMKIIalpha is secondary to this volume change. In addition, cLTP results
in a dramatic increase in the bound fraction of GFP-CaMKIIalpha in spines. To further study the bound
pool, immunogold electron microscopy was used to measure CaMKIIalpha in the pos tsynaptic density
(PSD), an important regulator of synaptic function. cLTP produced a persistent increase in the
PSD-associated pool of CaMKIIalpha. These results are consistent with the hypothesis that CaMKIIalpha
accumulation at synapses is a memory trace of past synaptic activity.
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June 18
2. Probing membrane dynamics in neuronal synapses
ML Renner (1), L Cognet (1,2), B Lounis (2) and D Choquet (1)
1) CNRS UMR 5091 University of Bordeaux-2, Bordeaux, France
2) CNRS UMR 5798 University of Bordeaux-1, Talence, France
Recent studies have shown that neurotransmitter receptors exchange between synaptic and
extrasynaptic spaces by lateral diffusion, being mobile also inside synapses. Their diffusion might play
an important role in synaptic transmission as the modification of synaptic strength depends on changing
the amount of synaptic glutamate receptors. We use single molecule and single particle techniques to
study the properties of the diffusing space inside synapses, analyzing the diffusion of two lipids and
AMPA receptor subunit GluR2. The lipids chosen were Gm1 (visualized by cholera toxin, ChTx) and
dioleoyl-phosphatidylethanolamine (DOPE). Outside synapses, DOPE showed anomalous or free
diffusion while GluR2 and ChTx showed confined or anomalous diffusion. Inside synapses all the
molecules are confined. This behavior could be changed by the disruption of the cytoskeleton, as after
F-actin depolymerization by latrunculin A, ChTx and DOPE showed free diffusion. In case of Glu R2,
latrunculin treatment causes a transient increase of mobility and a major loss of receptors in synapses.
The remaining synaptic receptors have reduced mobility, suggesting that cytoskeleton disruption allows
the mobile fraction to escape. In conclusion, synapses are a structured space that allows rapid although
confined diffusion and cytoskeleton integrity is important for this confinement.
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June 18
3. Voltage-sensitive dye imaging in vivo revealed a novel functional structure in monkey IT cortex
Ryota Homma and Manabu Tanifuji
RIKEN Brain Science Institute, Japan
We applied the technique of in vivo voltage-sensitive dye imaging to inferior temporal (IT) cortex
(area TE) of anesthetized macaque monkeys. The IT cortex is a visual association cortex that is involved
in object recognition. Past studies using intrinsic signal imaging and electrophysiology revealed that a
visual object is represented in IT cortex as a combination of spotty activities. Each spot has a size of a
cortical column (~0.5 mm) and reflects the spiking activity of neurons in the spot. In present study, we
demonstrate that the spatial pattern of synaptic potentials is different from that of spiking activity by
using voltage-sensitive dye imaging that allows us to record population averaged membrane potential
which emphasizing synaptic potentials. We stained the IT cortex with a voltage-sensitive dye RH-1692
and recorded the neural responses to visual stimuli presentation. We observed that a depolarization was
evoked by an object image. Several remarkable characteristics were noticed about the spatial pattern of
this visually evoked depolarization. (1) The depolarization was not localized into an area of columnar
size but spread over larger area that could include multiple columns. (2) The spatial patterns were
different from stimulus to stimulus presented. (3) However, an area of several square millimeters was
commonly activated by a group of different stimuli. Thus synaptic inputs to the IT cortex are given
not only to the sites showing strong spiking activity but also to the surrounding region of those sites.
Characteristics of the spatial pattern of synaptic potentials might imply the presence of a novel functional
structure in IT cortex that has a larger size than the size of cortical column and is specific to synaptic
potentials.
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June 20
1. Presynaptic Ca2+ signalling at individual mossy fibre synapses
Ricardo Scott and Dmitri Rusakov
Institute of Neurology, UCL, London, UK
Synaptic transmission between mossy fibres (MFs) and CA3 pyramidal cells exhibits profound
use-dependent facilitation and a distinct, NMDA receptor independent, mechanism of LTP. The
underlying molecular machinery, including mechanisms of presynaptic Ca2+ signalling, remains subject
of intense debate. Here we employ fast two-photon microscopy to examine rapid Ca2+ kinetics at
individual, giant MF boutons (GMFBs) imaged in area CA3 at a distance of up to 1.2 mm from the
dentate granule cell body (held in whole cell mode). We use double fluorescent labelling and several Ca2+
imaging protocols to assess that: (i) endogenous Ca2+ buffering is subject to partial washout in the
proximal varicosities (100 Нm from the soma), but not in the (distant) GMFBs, on the scale of hours; (ii)
distant, but not proximal, axonal segments appear electrically isolated from the soma; (iii) according to
preliminary estimates, an action potential drives a ~50 НM increment of total Ca 2+ inside a GMFB
(yielding a ~370 nM transient followed by a ~160 nM residual increase of free Ca 2+ from the resting
level of ~200 nM); and (iv) partial saturation of endogenous Ca2+ buffer during repetitive spikes could
fully explain use-dependent facilitation at MF - CA3 pyramidal cell synapses.
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June 20
2. The origin of synaptically-evoked Ca2+ transients in hippocampal dendritic spines
Ryosuke Enoki and Alan Fine
National Institute for Medical Research, London, UK
The generation of localized Ca2+ transients in dendritic spines is important because of the diverse and
crucial signaling functions of Ca2+ within nerve cells; the mechanisms underlying these transients can be
expected to have a major influence on synaptic plasticity in development and learning. The source of
Ca2+ during these transients has been highly controversial. We previously reported that single synaptic
events evoked NMDA receptor (NMDAR)-dependent Ca2+ release from ryanodine-sensitive internal
stores within spines at associational (Emptage et al., 1999) but not mossy fibre (Reid et al., 2001)
synapses; other groups, however, ruled out a role for internal stores (e.g., Kovalchuk et al., 2000). We
also reported that AMPA receptor (AMPAR) activation is necessary to remove Mg2+ block from
NMDARs at resting membrane potentials and that Ca2+ influx via the NMDARs is small and barely
detectable but essential for triggering the larger store release, whereas other groups reported that AMPAR
activation is not necessary and Ca2+ influx through NMDARs is the main source of the detectable Ca2+
transient (Koester and Sakmann, 1999; Kovalchuk et al., 2000). The basis of these differences has been
unclear. Here, using confocal microscopy in conjunction with intracellular recording in hippocampal
pyramidal cells, we demonstrate that ryanodine-sensitive store release is indeed the major source of
synaptically-evoked Ca2+ transients in associational, but not mossy fibre, spines. This store function is
rapidly lost, however, when recording is carried out via whole-cell patch pipettes. We also show that
putative synaptic stimulation via local stimulating electrodes (within 30 m of the spine) artificially
enhances NMDAR activation and overcomes the physiological requirement for AMPAR activation, most
likely by direct postsynaptic depolarization. Previous studies of neuronal Ca2+ dynamics carried out with
whole-cell recording have thus underestimated the importance of ryanodine receptor-mediated store
release, which provides an important amplification mechanism that may also serve to integrate
convergent biochemical signals.
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June 20
3. Correlation between optically recorded Ca influx in one presynaptic terminal and amplitude of
PSP in identified synapse in vivo.
Balaban P.M., Malyshev A.Yu
Institute of. Higher Nervous Activity and Neurophysiology, Moscow, Russia
Using the fast CCD camera (Redshirtimaging Ltd, USA) we recorded free Ca transients from the
neuropile in a preparation of isolated nervous system of terrestrial snail Helix lucorum in which one
identified presynaptic mechanosensory neuron was pressure injected with the Ca-sensitive dye Oregon
Green 488 BAPTA 1. During the experiment one intracellular electrode was inserted in stained
presynaptic mechanosensory cell, and the second one in identified postsynaptic withdrawal interneuron.
It was possible to record Ca from branches of sensory cell in neuropile where the processes of the stained
neurons are visible. In the neuropile were observed small, occupying just 1-3 pixels, varicosities of the
stained neuron branches that show strong signal-to-noise responses to an action potential induced in
soma. Location of those areas in neuropile, and significantly bigger (than in the processes) Ca increase
during spike generation suggests that those varicosities may be presynaptic terminals. We compared
changes in amplitude of the Ca transients in those putative terminals with the amplitude of postsynaptic
responses to one presynaptic spike recorded intracellularly. It was observed that serotonin bath
application elicited no changes in membrane potential of postsynaptic neuron, but elicited significant
changes in presynaptic Ca influx during spike generation that coincided with changes in amplitude of
postsynaptic potential. Blockade of cannabinoid receptors effectively increased the EPSPs amplitude and
Ca influx in presynaptic terminal, but only after preliminary tetanization of the postsynaptic cell. Role of
cannabinoids in regulation of synaptic plasticity is discussed. Supported by Russian Foundation for Basic
Research, CRDF grant RB1-2321-MO-02.
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POSTER ABSTRACTS
June 19
1. Reactive oxygen species are involved in modulation of neuronal activity by ATP in neonatal rats
Ramil Afzalov
University of Helsinki, Finland
A growing body of evidence suggests that reactive oxygen species (ROS) are important signaling
molecules in the CNS. In the present study, we tested the hypothesis that, in immature brain, one of the
widely distributed neurotransmitters ATP operates via production of ROS. In neonatal rats and mice, in
pyramidal cells of CA3 region of hippocampus, ATP increased the frequency of spontaneous
postsynaptic potentials (SPSPs) and decreased the frequency of giant depolarizing potentials (GDPs), an
immature brain-specific type of neuronal network activity. In the presence of DPCPX which is an
antagonist of adenosine receptors, ATP still increased the frequency of SPSPs via P2Y1 receptors and
bi-directionally changed (first increased then decreased) the frequency of GDPs via PPADS-sensitive P2
receptors. The effect of ATP on SPSPs (but not on GDPs) was eliminated by N-acetylcysteine, a strong
antioxidant. For a series of imaging experiments in immature hippocampal slices, w e used
dichlorodihydrofluorescein (DCF) as a fluorescent probe for ROS. DCF was constantly present in bath to
compensate for its leak from cells and to minimize artifacts caused by photoactivation and
photobleaching of the dye. ATP induced production of ROS and increased the frequency of Ca 2+
oscillations detected by fluo-4 loaded into the cells by using its AM ester form. The exogenous oxidant,
H2O2 in low physiological concentrations did not affect Ca2+ oscillations but reproduced the ATP-induced
increase in DCF fluorescence intensity. H2O2 also mimicked the facilitatory effects of ATP on GDPs and
SPSPs which showed similar sensitivity to N-acetylcysteine. Taken together our data suggest that in
immature brain ATP produces (via P2Y1 receptor mediated excitation of astrocytes) ROS which in turn
modulate GABAergic inputs to interneurons.
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2. Synapse-to-synapse variation of calcium channel subtype compositions in large mossy fiber
terminals of mouse hippocampus
Kenichi Miyazaki, Toru Ishizuka, Hiromu Yawo
Tohoku University, Grad Sch Life Sci, Sendai, Japan
Both N-and P/Q-type voltage-dependent calcium channels (VDCCs) are involved in the fast
transmitter release in the hippocampus. Although the variable compositions of presynaptic VDCC
subtypes have been suggested to give a neural network of huge complexity and flexibility, their presence
has been only demonstrated at presynaptic terminals of cultured neurons and has remained unclear in the
brain. We have previously shown that at least four VDCC subtypes N-, P/Q-, R- and L-type are present in
the mossy fiber (MF) terminals. Here, the composition of presynaptic VDCC subtypes were directly
investigated on the individual large mossy fiber synapses in the hippocampal slice of mouse. The
strontium influx was fluorometrically measured from a single large MF terminal applied with Oregon
Green 488 BAPTA-1 dextran, a fluorescent indicator of Sr(2+) and Alexa Fluor 546 conjugated dextran,
an indicator of structure under conventional confocal microscopy. The composition of presynaptic
VDCC subtypes were investigated using subtype-selective neurotoxins, omega-conotoxin GVIA (an
N-type selective blocker), omega-agatoxin IVA (a P/Q-type selective blocker) and SNX-482 (an R-type
selective blocker). We found that individual large MF terminals were unique in their fraction of N-type
VDCCs in the acute slice of mouse hippocampus. In contrast, the fraction of P/Q-type VDCCs was rather
uniform. Furthermore neighboring large presynaptic terminals on the same axon are similar in their
P/Q-type fractions of VDCCs, but not in N- and R-type fractions. These results provide direct evidence
that large MF synapses are non-uniform in the composition of presynaptic N- and R-type VDCC
subtypes. We suggest that the synapse-to-synapse variation of presynaptic VDCC subtypes give a MF
network huge complexity that is temporally variable.
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3. Activity-dependent BDNF exocytosis at mossy fiber terminals of mouse hippocampus
Suyama Shigetomo, Ishizuka Toru, Yawo Hiromu
Tohoku University, Grad Sch Life Sci, Sendai, Japan
Brain-derived neurotrophic factor (BDNF) was first identified as a molecule regulating neuronal
survival and differentiation, but it also has a pivotal role in the regulation of synaptic strength. It is
known that BDNF is released from postsynaptic neurons as one of retrograde or paracrine messengers.
However, recent studies have shown that BDNF is also anterogradely transported from soma to axon and
stored in the presynaptic terminals. The mossy fiber (MF) terminals in hippocampus contain the highest
concentration of BDNF in the CNS. These observations led us to hypothesize that BDNF is released
from presynaptic terminals, especially the giant boutons of MFs in an activity-dependent manner. Here
we report that BDNF is released from the giant mossy fiber boutons by the activity-dependent manner.
We made a sindbis virus vector containing a mRNA coding the fusion protein construct of BDNF and
Venus@(BDNF-Venus), one of green fluorescent protein derivatives, and inoculated it stereotaxically to
the dentate granule cells of mouse hippocampus (P14-21). After 2-3 days, MF boutons accumulating
BDNF-Venus were identified in the acute slice under confocal microscopy. The fluorescence was
measured from a single giant MF terminal, and was decreased in intensity by the high-frequency
stimulation applied on the MF axon. This response depended on extracellular calcium, and disappeared
by either removing extracellular calcium or treating with N-methylmaleimide (NEM). These
observations would provide first and direct evidences that BDNF is actually released from the giant MF
boutons through activity-dependent exocytosis.
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4. Cross talk between metabotropic and ionotropic glutamate receptor in parallel fiber-induced
inositol 1,4,5-trisphosphate production in cerebellar Purkinje cells
Yohei Okubo, Sho Kakizawa, Kenzo Hirose and Masamitsu Iino
University of Tokyo, Japan
In many excitatory glutamatergic synapses, both ionotropic glutamate receptors (iGluRs) and
metabotropic glutamate receptors (mGluRs) are closely distributed on the postsynaptic
membrane. However, the functional significance of the close distribution of the two types of glutamate
receptors has not been fully clarified. In this study, we examined the functional interaction between
iGluR and mGluR at parallel fiber (PF)-Purkinje cell synapses in the generation of inositol
1,4,5-trisphosphate (IP3), a key second messenger that regulates many important cellular functions. We
visualized local IP3 dynamics in Purkinje cells using the GFP-tagged pleckstrin homology domain
(GFP-PHD) as a fluorescent IP3 probe. Purkinje cells were transduced with sindbis virus encoding
GFP-PHD and imaged with a two-photon laser scanning microscope. Translocation of GFP-PHD from
the plasma membrane to the cytoplasm due to an increase in IP3 concentration was observed upon PF
stimulation in fine dendrites of Purkinje cells. Surprisingly, this PF-induced IP3 production was blocked
not only by group I mGluR antagonist but also by the AMPA receptor (AMPAR) antagonist. The
PF-induced IP3 production was blocked by either the inhibition of G-protein activation by GDP-bS or
intracellular Ca2+ buffering by BAPTA. These results show that IP3 production is mediated
cooperatively by group I mGluR and AMPAR through G-protein activation and Ca2+ influx at
PF-Purkinje cell synapses, identifying the robust cross talk between iGluR and mGluR for the generation
of IP3 signals.
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5. Optical measurement of exocytosis using synapto-pHluorin transgenic mouse
Rikita Araki, Hiroyuki Sakagami, Yuchio Yanagawa, Takuya Hikima, Toru Ishzuka, Hiromu Yawo
Tohoku University, Sendai, Japan
Hippocampal mossy fiber synapses are known to exhibit plasticity in the presynaptic terminals, e.g.
frequency facilitation and presynaptic form of long-term potentiation (LTP). To further investigate the
detailed mechanisms of the presynaptic plasticity, we have generated the synapto-pHluorin(SpH)
transgenic mouse lines. In these transgenic lines, the expression of SpH was regulated by the neuron
specific Thy-1.2 promoter or Cre / loxP recombination system. In Thy-1.2 regulated SpH mouse, SpH
was expressed in broad spectrum of regions of the brain (4 lines), or specific regions as hippocampus and
cerebellum (2 lines). In one of them, SpH expression was specific in the mossy fiber terminals in the
hippocampus. On the other hand, Cre / loxP regulated SpH mouse was crossed to some mouse lines
expressing cre recombinase in specific types of the cell, and SpH was expressed in the corresponded
axonal region in each offspring. In acute brain slice from these mice, individual presynaptic terminals
were easily identified by the SpH fluorescence under the confocal microscopy. The SpH fluorescence
intensity was somewhat decreased by acidic buffer surperfusion and greatly increased by vesicular
neutralization of pH, indicating that they are mainly distributed in the synaptic vesicles. Electrical
stimulation at the hippocampal mossy fiber elicited the transient increase of fluorescent intensity at
individual presynaptic terminals, and the decline phase of these changes was blocked by perfusion of
bafilomycin A1, an inhibitor of the vesicular proton pump. We are going to characterize the mode of
presynaptic exocytosis during the mossy fiber LTP which is not dependent on the postsynaptic NMDA
receptors. These transgenic lines are suggested to be useful for the morphological and physiological
studies of presynaptic terminals and also for the in vivo study.
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6. Imaging early phase of synaptogenesis: implication for the role of postsynaptic protrusion in
target recognition of Drosophila neuromuscular junction
Hiroshi Kohsaka, Etsuko Takasu, Akinao Nose
University of Tokyo, Japan
Synaptogenesis occurs between pre- and postsynaptic cells. While a growth cone of a presynaptic
cell shows high maneuverability, a postsynaptic cell also extends motile filopodia such as dendritic
filopodia of neuron or myopodia of muscle. Though the period of emergence of these postsynaptic
protrusions is limited in early phase of synaptogenesis, the role(s) of them are still unclear. In this study,
we imaged and analyzed muscular protrusion (myopodia) of Drosophila embryos during early phase of
neuromuscular synaptogenesis using confocal microscopy. We analyzed the distribution of three kinds
of YFP-tagged protein in myopodia of muscle 12 (M12): target recognition molecule Capricious (Caps),
which is expressed in subsets of muscles and determines the target specificity of neuromuscular
connectivity of M12, cell adhesion molecule Fasciclin II (FasII), and scaffolding protein Discs Large
(Dlg). Before initial interaction between myopodia and growth cones, Caps-YFP strongly accumulated
at the tips of myopodia. On the other hand, FasII-YFP and Dlg-YFP are distributed uniformly along
myopodia. This elaborate regulation of protein distribution in myopodia implies that myopodia might
have active roles in target recognition. To examine the dynamics of myopodia in target recognition
process, we performed in vivo time-lapse imaging of myopodia and motoneuronal growth cones using
membrane -bound form GFP. We could observe contacts between the tips of myopodia and growth
cones in vivo. While about half of the contacts remained during synaptogenesis, the other half was
unstabilized and drew apart. In addition, most of contacts between mismatched partner cells are
unstable. These observations imply that the role(s) of postsynaptic protrusions are not only expanding
the surface area of target cells but also providing information about the target identity toward growth
cones.
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7. Imaging retinotectal arborisation and synaptogenesis in vivo in the zebrafish: novel roles for
robo-slit signalling
Douglas S. Campbell, Tong Xiao, Herwig Baier and Chi-Bin Chien
University of Utah, Salt Lake City, USA
During the development of the zebrafish retinotectal projection, retinal ganglion cell (RGC) growth
cones navigate along a highly stereotypical pathway from the retina to their primary target, the optic
tectum in the midbrain. Once in the tectum, growth cones undergo arborisation and synaptogenesis. The
molecular mechanisms underlying these processes in the CNS are largely unknown. To study the
development and molecular mechanisms underlying arborisation and synaptogenesis, we are using
laser-scanning confocal microscopy to visualise single RGC axons and their arbors in vivo in living
zebrafish embryos, after labelling with GFP fusion proteins under the control of an RGC-specific
promoter. We examined wild-type arbors between 76 and 100 hours post fertilisation (hpf) and observed
a stereotyped increase in branch tip number and arbor area during this period. Using in situ hybridisation
to characterise the expression of zebrafish roundabout homologues (robos) and their presumptive ligands,
the slits, at 76 and 100hpf, we found that slit1a is expressed in the tectum, while robo2 is expressed in
RGCs and the tectum. This suggests that Robo-Slit signalling may be involved in RGC arborisation. To
test this hypothesis, we observed RGC arbors in astray (robo2) mutant embryos and in embryos where
slit1a protein had been knocked-down by injection of antisense morpholinos. RGC arbors from astray
mutants and slit1a morphants have approximately double the number of branch tips, total arbor lengths
and arbor area as age-matched wild-type arbors. Labelling of presumptive RGC synapses with a
vesicle-associated membrane protein-GFP (VAMP-GFP) or Rab3-YFP fusion proteins revealed a similar
increase in numbers of puncta in arbors from astray mutant embryos, suggesting an increase in synapse
number compared to wild-type arbors. These observations suggest that robo2 and slit1a are negative
regulators of zebrafish RGC arborisation and synaptogenesis in vivo, in contrast to the previously
described branch-inducing effects of Slit in vitro.
37