Department ofIntegrative Physiology
Outline and Research Objectives
This department was initially established in 1877 as the First Department of Physiology,
and reorganized in 1997 as the Department of Integrative Physiology. Our department collaborates with other laboratories dedicated to Physiological Sciences, that is, theDepartment of Molecular/Cellular Physiology and the Department of Neurophysiology, inteaching activities for undergraduate courses and the nursing school. The fields in which
our department specializes include the entire spectrum of the physiology of “animal functions”, such as general physiology, sensory physiology, neurophysiology, higher nervous
functions and cognitive neurosciences.

Faculties and Students
Professor and Chair Yasushi Miyashita, Ph.D. (1989- )Lecturer
Isao Hasegawa, M.D., Ph.D. (2000- )Associate .................................2
Postdoctoral Fellow ................5
Graduate Student..................10
Research Student ....................1
Secretary .................................3

lus-selectivity of IT neurons can be acquired throughlearning in adulthood. Second, the activity of IT neurons can link the representations of temporally associated but geometrically unrelated stimuli.

Most of our research has been focused on thehigher brain functions of the mammalian central nervous system, in particular, the neural mechanisms of
cognitive memory in the primate. The basic motivation of the research can be best explained as follows:
Knowledge or experiences are voluntarily recalledfrom memory by reactivation of their neural representations in the cerebral association cortex. Three questions are central in the understanding of this process:(1) Where are mnemonic representations coded andhow are they organized?
(2) Which neural processes create the representation?
(3) What is the mechanism underlying reactivation of
the representation on demand of voluntary recall?

2. Activating the representation in the temporal
cortex on demand
In spite of the classical clinical observation thatelectric stimulation of the temporal lobe produces‘experiential responses’, there have been no direct evidence supporting the notion of ‘reactivation of neuralrepresentations’ during memory retrieval. We firstreported a neuronal correlate of the reactivation
process as ‘pair-recall neurons’ [42, 45]. In our subsequent study [30], we devised a new modified pairassociate task in which the necessity for memory
retrieval and its initiation time were controlled by acolour switch, independent of the cue stimulus presentation. Single-unit recordings in monkeys performing this task revealed that IT neurons selective to amemorized object are dynamically activated at thetime of memory retrieval of that object, and suppressed at the time of retrieval of other objects. Then,it became important to determine the neural networkthat drives the memory retrieval machinery in the IT
cortex.

1. Creating the mnemonic representation
Lesion studies in primates have implicated the IT
cortex in long-term memory storage of visual objects[40]. Neuronal correlates of associative long-term
memory were first discovered in the IT cortex byMiyashita [49, 50] and Sakai & Miyashita [45]. Hedeveloped a novel memory paradigm that requires the
subject to create a link of associative memory
between mathematically designed pictures (for examples, see Fig.1). Their single-unit recording experiments in thepair-association task identified two
mnemonic properties of IT neurons. First, the stimu-

Figure 1 : Paired associates. Each pictures was created according to a fractal algorithm.

Past Research and Major Accomplishments30

Figure 2 : Which information is carried via top-down signals? (a) A stimulus-stimulus association task. Twenty cue-pictures are randomly sorted into five categories. Each of the four cues in one category specifies a common choice. (b) Category-selective delay
activity of an inferior temporal neuron. Delay activities were raised for all cues in Category I, but not for any cues in Category V(rastergrams, top-down condition). Choice responses are also strongest for Category I and weakest for Category V. Spike density
functions show averaged activities across four cues in Category I (thick) and in Category V (thin) for both conditions (top-down,
blue; bottom-up, black). (c) Spike density functions of the top-down response for five categories, as well as the choice responses,
shown by a pseudo-colour coding. Note that the category-selective delay activity can predict choice selection.

3. Top-down activation through fronto-temporalpathway
A candidate component of the neural circuit is the
top-down activation from the prefrontal cortex. The
prefrontal cortex has been implicated in various executive processes, and its contribution to mnemonic
functions, particularly in episodic memory and working memory, is repeatedly demonstrated in humanneuroimaging studies. We attempted to directly test
its contribution to memory retrieval control by thecapacity for interhemispheric transfer of mnemonic
signal through the anterior corpus callosum, a key
structure interconnecting prefrontal cortices. We
introduced the posterior-split-brain paradigm into theassociative memory task in monkeys [23]. Long-term
memory acquired through stimulus-stimulus association did not interhemispherically transfer via theanterior corpus callosum. Nonetheless, when a visualcue was presented to one hemisphere, the anteriorcallosum could instruct the other hemisphere to
retrieve the correct stimulus specified by thecue. Therefore, although visual long-term memory is
stored in the temporal cortex, memory retrieval isunder the executive control of the prefrontal cortex.
In spite of predictions based on these behaviouralexperiments, no neuronal correlate of the top-down
signal from the prefrontal cortex to IT cortex had
been detected. We provided the first evidence of theexistence of the top-down signal [14] by conducting
single-unit recording in posterior-split-brainmonkeys. In the absence of bottom-up visual inputs,
single IT neurons were robustly activated by the topdown signal, which conveyed information on semantic categorization imposed by visual stimulus-stimulus
association (Fig. 2). We also demonstrated that the

top-down signal had a longer latency by about 100 ms
than the bottom-up signal. The longer latency is most
likely ascribed to a multi-synaptic conduction delaythat reflected the signal transformation within theprefrontal cortices.

Current Research and Future Prospects
Most of our current research is focused on the third
question on the mechanisms of cognitive memory, thatis, (3) What is the mechanism underlying reactivationof the representation on demand of voluntary recall?
Two complementary approaches are of interest in
answering the above question. First, neuroimaging
studies in humans can elucidate brain representations
and their interactions in high-level memory processes,
which have been conceptualized as retrieval attempt
or contextual monitoring. Second, the well-analyzed
neural representations in the monkey cortex should
be compared with those in the human cortex. The
comparison would provide strong evidence that couples active neural codes in the brain network withconscious experiences.
An example of our recent achievements along thefirst approach is the discovery of a neural correlate of
the “feeling-of-knowing” [1]. The “feeling-of-knowing(FOK)” is a subjective sense of knowing a word before
recalling it, and it provides us clues to understanding
the mechanisms of human meta-memory systems.
We investigated neural correlates for the FOK basedon the recall-judgment-recognition paradigm. Eventrelated functional magnetic resonance imaging (fMRI)
with parametric analysis was used. We found activations in the left dorsolateral, left anterior, bilateralinferior, and medial prefrontal cortices that signifi-

31

Figure 3 : Comparison of activated cortical areas in set-shifting of WCST in monkeys (left) and humans (right).

cantly increased as the FOK became greater.
Furthermore, we demonstrated that the FOK-region
in the right inferior frontal gyrus and a subset of the
FOK-region in the left inferior frontal gyrus are notrecruited for successful recall processes, suggesting
their particular role in meta-memory processing.
Along the second approach, we have recently
demonstrated that fMRI studies in macaque monkeys
allow us to directly compare the brain activity ofhumans with that of monkeys with respect to highlevel cognitive functions [2]. In this study, the functional brain organization of macaque monkeys andhumans was directly compared by fMRI. Subjects ofboth species performed a modified Wisconsin Card
Sorting Test that required behavioural flexibility inthe form of cognitive set shifting. Equivalent visual
stimuli and task sequence were used for the two
species. We found transient activation related to cognitive set shifting in focal regions of the prefrontal
cortex in both monkeys and humans. These functional homologs were located in cytoarchitectonicallyequivalent regions in the posterior part of the ventrolateral prefrontal cortex. This comparative imaging
provides insights into the evolution of cognition inprimates.
Various neuroimaging studies have been carriedout to clarify cognitive functions of the humanbrain. Because most neuroimaging studies rely on thecorrelation between cognitive processes and brain
activations, conjunction with other complementary
methods, such as neuropsychological studies andtranscranial magnetic stimulation, should be promoted to clarify the behavioural significance of observed
brain activities. Emerging fMRI of the monkey brain
enables us to directly compare the brain activity ofhumans with that of monkeys using the same modality. Therefore the monkey fMRI will make it possible
to combine imaging studies with electrophysiology orlesion studies, and will hopefully lead to the understanding of the causal relationship between activatedbrain areas and cognitive functions. Since most of thedetailed knowledge of anatomy and function of thecerebral cortex has come from studies in monkeys,
sharing the same method among the studies of monkeys and humans would advance the understandingof the neural basis of human cognition.

32

Research Grants
1. A Grant-in-Ai32d for Specially Promoted Research,
MEXT (07102006)
Neural mechanisms of cognitive memory system :Integration of functional magnetic resonance imaging method and molecular/cellular approaches.
¥ 215,000,000 (1995-1999)
2. A Grant-in-Aid for Specially Promoted Research,
MEXT (Extension 07102006)
Neural mechanisms of cognitive memory system :Integration of functional magnetic resonance imaging method and molecular/cellular approaches.
¥309,000,000 (2000-2001)
3. A Grant-in-Aid for Specially Promoted Research,
MEXT (14002005)
Brain distributed network : Integrative study basedon functional MRI in monkeys¥ 550,000,000 (2002-2006)

Select Publications
1. Kikyo, H., Ohki, K. and Miyashita, Y. Neural correlates for “feeling-of-knowing”: an fMRI parametricanalysis.
Neuron 36, 177-186, 2002.
2. Nakahara, K., Hayashi, T.,Konishi, S. and Miyashita,
Y. Functional MRI of macaque monkeys performinga cognitive set-shifting task.
Science 295, 1532-1536, 2002.
3. Konishi, S., Hayashi, T., Uchida, I., Kikyo, H.
Takahashi, E. and Miyashita, Y. Hemispheric asymmetry in human lateral frontal cortex during congnitive set shifting.
Proc. Natl. Acad. Sci. USA 99, 7803-7808, 2002.
4. Hasegawa, I. and Miyashita, Y. Making categories:
expert neurons look into key features.
Nature Neurosci. 5, 90-91, 2002.
5. Tokuyama, W., Okuno, H., Hashimoto, T., Li, X.Y.
and Miyashita, Y. Selective zif268 mRNA inductionin the perirhinal cortex macaque of monkeys duringformation of visual pair-association memory.
J. Neurochem. 81, 60-70, 2002.
6. Matsuzaki, M., Ellis-Davies, G.C.R., Nemoto, T.,
Miyashita, Y., Iino, M. and Kasai, H. Dendritic spine
geometry is critical for AMPA receptors expression
in hippocampal CA1 Pyramidal neurons.
Nature Neurosci. 4, 1086-1092, 2001.

7. Naya, Y., Yoshida, M. & Miyashita, Y. Backward
spreading of memory retrieval signal in the primate
temporal cortex.
Science 291, 661-664, 2001.
8. Li, X.Y., Hashimoto, T., Tokuyama, W., Miyashita,
Y. and Okuno H Spatio-temporal dynamics of BDNF
mRNA induction in the vestibulo-olivary network
during vestibular compensation.
J. Neurosci 21, 2738-2748, 2001.
9. Miyashita,Y. and Farah, M.J. Cognitive neuroscienceat the turn of the millennium.
Curr. Opin. Neurobiol. 11, 147-149, 2001.
10. Nemoto, T., Kimura, R., Ito,K., Tachikawa, A.,
Miyashita, Y., Iino, M. and Kasai, H. Sequential
replenishment mechanism of exocytosis in pancreatic acini.
Nature Cell Biol. 3, 253-258, 2001.
11. Tokuyama,W., Okuno,H., Hashimoto, T., Li, Y.X.
and Miyashita, Y. BDNF upregulation during declarative memory formation in monkey inferior temporal
cortex.
Nature Neurosci.3, 1134-1142, 2000.
12. Embick, D., Marantz, A., Miyashita, Y., O’Neil W.
and Sakai, K.L. A syntactic specialization for Broca’s
area.
Proc. Natl. Acad. Sci. USA 97, 6150-6154, 2000.
13. Miyashita, Y. and Hayashi, T. Neural representationof visual objects : encoding and top-down activation.
Curr. Opin. Neurobiol. 10, 187-194, 2000.
14. Tomita, H., Ohbayashi, M., Nakahara, K., Hasegawa,I. and Miyashita, Y. Top-down signal originatingfrom the prefrontal cortex for memory retrieval.
Nature 401, 699-703, 1999.
15. Maeda, H., Ellis-Davies, G.C.R., Ito, K., Miyashita, Y.
and Kasai, H. Supralinear Ca2+ signaling by cooperative and mobile Ca2+ buffering in Purkinje neurons.
Neuron 24, 989-1002, 1999.
16. Kasai, H., Kishimoto, T., Liu, T., Miyashita, Y.,
Podini, P., Grohovaz, F. and Meldolesi, J. Multiple
and diverse forms of regulated exocytosis in wildtype and defective PC12 cells.
Proc. Natl. Acad. Sci. USA 96, 945-949, 1999.
17. Konishi, S., Nakajima, K., Uchida, I., Kikyo, H.,
Kameyama, M. and Miyashita, Y. Common inhibitory mechanism in human inferior prefrontal cortex
revealed by event-related functional MRI.
Brain 122, 981-991, 1999.
18. Konishi, S., Kawazu, M., Uchida, I., Kikyo. H.,
Asakura, I. and Miyashita, Y. Contribution of working memory to transient activation in human inferior
prefrontal cortex during performance of Wisconsin
card sorting test.
Cerebral Cortex 9, 745-753, 1999.
19. Okuno, H., Tokuyama, W., Li, Y-X., Hashimoto, T.
and Miyashita, Y. Quantitative evaluation of neurotrophin and trk mRNA expression in visual andlimbic areas along the occipito-temporo-hippocampal
pathway in adult macaque monkeys.
J. Comp. Neurol. 408, 378-398, 1999.

20. Uchida, I., Kikyo, H., Nakajima, K., Sekihara, K. andMiyashita, Y. Activation of lateral extrastriate areas
during orthographic processing of Japanese characters studied with fMRI.
Neuroimage 9, 208-215, 1999.
21. Ito, K., Miyashita, Y. and Kasai H Kinetic control ofmultiple forms of Ca2+ spikes by inositol trisphosphate in pancreatic acinar cells.
J. Cell Biol. 146, 405-413, 1999.
22. Takahashi, N., Kadowaki, T., Yazaki, Y., Ellis-Davies,
G.C.R., Miyashita, Y. and Kasai, H. Post-priming
actions of ATP on Ca2+-dependent exocytosis in pancreatic beta cells.
Proc. Natl. Acad. Sci. USA 96, 760-765, 1999.
23. Hasegawa, I., Fukushima, T., Ihara, T. andMiyashita, Y. Callosal window between prefrontal
cortices : cognitive interaction to retrieve long-term
memory.
Science 281, 814-818, 1998.
24. Konishi, S., Nakajima, K., Uchida, I., Kameyama, M.,
Nakahara, K., Sekihara K. and Miyashita, Y.
Transient activation of inferior prefrontal cortex during cognitive set shifting.
Nature Neurosci.1, 80-84, 1998.
25. Konishi, S., Nakajima, K., Uchida, I., Sekihara, K. andMiyashita, Y. Temporally resolved no-go dominant
brain activity in the prefrontal cortex revealed byfubctional magnetic resonance imaging.
Neuroimage 5, 120, 1997.
26. Takahashi, N., Kadowaki, T., Yazaki, Y., Miyashita,
Y. and Kasai, H. Multiple exocytotic pathways inpancreatic β cells.
J. Cell Biol. 138, 55-64, 1997.
27. Ninomiya, Y., Kishimito, T., Yamazawa, T., Ikeda,
H., Miyashita, Y. and Kasai, H. Kinetic diversity inthe fusion of exocytotic vesicles.
EMBO J. 16, 929-934, 1997.
28. Ito, K., Miyashita, Y. and Kasai H. Micromolar andsubmicromolar Ca2+ spikes regulating distinct cellularfunctions in pancreatic acinar cells.
EMBO J. 16, 242-251, 1997.
29. Higuchi, S. and Miyashita, Y. Formation of mnemonic neuronal responses to visual paired associates ininferotemporal cortex is impaired by perirhinal andentorhinal lesions.
Proc. Natl. Acad. Sci. USA, 93, 739-743, 1996.
30. Naya, Y., Sakai, K. and Miyashita, Y. Activity of primate inferotemporal neurons related to a sought target in pair-association task.
Proc. Natl. Acad. Sci. USA, 93, 2664-2669, 1996.
31. Kasai, H., Takagi, H., Ninomiya, Y., Kishimoto, T.,
Ito, K., Yoshida, A., Yoshioka., T. and Miyashita, Y.
Two components of exocytosis and endocytosis inpheochromocytoma cells studied using caged-Ca2+
compounds.
J. Physiol. 494, 53-65, 1996.
32. Ninomiya, Y., Kishimoto, T., Miyashita, Y. andKasai, H. Ca2+-dependent exocytotic pathways in chinese hamster ovary fibroblasts revealed by a cagedCa2+ compound.

33

J. Biol. Chem. 271, 17751-17754, 1996.
33. Sakai, K., Watanabe, E., Onodera, Y., Uchida, I.,
Kato, H., Yamamoto, E., Koizumi, H. and Miyashita,
Y. Functional mapping of the human colour centre
with echo-planar magnetic resonance imaging.
Proc. R. Soc. Lond. B 261, 89-98, 1995.
34. Miyashita, Y. : How the brain creates imagery:
Projection to primary visual cortex.
Science 268, 1719-1720, 1995.
35. Okuno, H., Saffen, D.W. and Miyashita, Y.
Subdivision-specific expression of Zif268 in the hippocampal formation of the Macaque monkey.
Neuroscience 66, 829-845, 1995.
36. Sakai, K., Watanabe, E., Onodera, Y., Itagaki, H.,
Yamamoto, E., Koizumi, H. and Miyashita, Y.
Functional mapping of the human somatosensory
cortex with echo-planar magnetic resonance imaging.
Magn. Reson. Med. 33, 736-743, 1995.
37. Sakai, K. and Miyashita, Y. Visual imagery : An interaction between memory retrieval and focal attention.
Trends Neurosci. 17, 287-289, 1994.
38. Mori, A., Takahashi, T., Miyashita, Y. and Kasai, H.
Two distinct glutamatergic synaptic inputs to thestriatal medium spiny neurones of neonatal rat andpaired-pulse depression.
J. Physiol. 476, 217-228, 1994.
39. Kasai, H., Li, Yeu Xin and Miyashita, Y. Subcellular
distribution of Ca2+ release channels underlying Ca2+
waves and oscillations in exocrine pancreas.
Cell 74, 669-677, 1993.
40. Miyashita, Y. Inferior temporal cortex: where visual
perception meets memory.
Annu. Rev. Neurosci. 16, 245-263, 1993.
41. Sakai, K. and Miyashita, Y. Memory and imagery in
the Temporal lobe.
Curr. Opin. Neurobiol. 3, 166-170, 1993
42. Miyashita, Y., Date, A. and Okuno, H.
Configurational encloding of complex visual forms
by single neurons of monkey temporal cortex.
Neuropsychologia 31, 1019-1031, 1993.
43. Maruyama, Y., Inooka, G., Li, Y., Miyashita, Y. andKasai, H. Agonist-induced locarized Ca2+ rises directly
triggering exocytotic secretion in exocrine pancreas.
EMBO J. 12, 3017-3022, 1993.
44. Iino, M., Yamazawa, T., Miyashita, Y., Endo, M. andKasai, H. Critical intracellular Ca2+ concentration for
all-or-none Ca 2+ spiking in single smooth muscle
cells.
EMBO J. 12, 5287-5291, 1993.
45. Sakai, K. and Miyashita, Y. Neural organization forthe long-term memory of paired associates.
Nature 354, 152-155, 1991.
46. Rolls, E.T., Miyashita, Y., Cahusac, P., Kesner, R.P.,
Niki, H., Feigenbaum, J. and Bach, L. Hippocampal
neurons in the monkey with activity related to theplace in which a stimulus is shown.
J. Neurosci. 9, 1835-1845, 1989.
47. Mori, K. and Miyashita, Y. Localized metabolic
responses to optokinetic stimulation in the brain

34

stem nuclei and the cerebellum investigated with the[14C]2-deoxyglucose method in rats.
Neuroscience 30, 271-281 1989.
48. Miyashita, Y., Rolls, E.T., Cahusac, P. and Niki, H.
Activity of hippocampal formation neurons in themonkey related to a stimulus-response association
task.
J. Neurophysiol. 61, 669-678, 1989.
49. Miyashita, Y. Neuronal correlate of visual associative
long-term memory in the primate temporal cortex.
Nature 335, 817-820, 1988.
50. Miyashita, Y. and Chang, H.S. Neuronal correlate of
pictorial short-term memory in the primate temporal
cortex.
Nature 331, 68-70, 1988.

Department of Cellular andMolecular Physiology
Outline and Research Objectives
Knowledge comes to man through the door of the senses (Heraclitus). Using multidisciplinary approaches including electrophysiology, optical imaging, molecular / cell biology, andmolecular genetics, we aim at better understanding of neuronal mechanisms for the sensory
perception of the external world and for the emotional state induced in the brain by thesensory inputs. For this purpose we are currently analyzing the central nervous system forolfaction, a sensory modality that has a strong influence to human emotion. Another major
research focus of this department is to understand cellular and molecular mechanisms forcontact-mediated interactions between neurons and immune cells that occur in pathologicaland physiological conditions.

Faculties and Students
Professor and Chair Kensaku Mori, Ph. D. (May 1999 ~)Lecturer
Masahiro Yamaguchi, M.D., Ph. D.
Associate .................................2
Graduate student ..................12
Research student.....................1
Secretary .................................1(e.g., telencephalin (TLCN), Yoshihara et al., Neuron,
1994; OCAM, Yoshihara et al., J. Neurosci., 1997). We
are currently extending the analysis of the functionalroles of these cell adhesion molecules using TLCNdeficient mice (in collaboration with Prof. Mishina)
and OCAM-deficient mice. During the course of theanalysis of TLCN, we unexpectedly found that thebinding between TLCN and its counterreceptor LFA-1
mediated the interaction between neurons andPast Research and Major Accomplishments
K. Mori and colleagues have been studying thefunctional organization of the olfactory nervous system for nearly 28 years. Major accomplishment of ourresearch group before 1999 includes (1) a systematicfunctional and structural analysis of the neuronal circuits in the mammalian olfactory bulb, the first centerfor the processing of olfactory information in thebrain, (2) the finding that individual principal neurons
in the olfactory bulb respond selectively to a range ofodorants having common molecular structures, and(3) the finding of zone-to-zone axonal connectivity
pattern between olfactory sensory epithelium and theolfactory bulb. After K. Mori’s arrival at the
Department of Cellular and Molecular Physiology in
May 1999, we further extended our previous studies
and found a clue for understanding the spatial organization of the ‘odor map’ in the olfactory bulb (Fig.1; e.
g., Mori et al., Science, 1999, Uchida et al., Nature
Neurosci., 2000., Nagao et al., NeuroReport, 2000).
These studies as well as our novel molecular genetic
approaches (Yoshihara et al., Neuron 1999) formed abasis for current and future studies on the ‘odor maps’
in the olfactory bulb and olfactory cortex (e.g., Inaki
et al., Eur. J. of Neurosci., 2002).
We previously found several novel neuronal celladhesion molecules and analyzed their functionalroles in the formation of neuronal circuit in the brain

35

immune cells (Mizuno et al., J. B. C., 1997; Tian et al.,J. Cell Biol., 2000, Eur. J. Immunol., 2000, J. Immunol.,1997). We thus developed a co-culture assay system to
examine the contact-mediated interactions between
neurons and immune cells.
Our group generated transgenic mice in which
newly generated neurons are visualized in the brain
by the green fluorescent protein (Yamaguchi et al.,NeuroReport, 2000). Using the transgenic mice, we are
now studying the neurogenesis and neuron-elimination in the adult brain.

Current Research
Current research programs and targets can be categorized into the following four topics.
(1) Functional analysis of the neuronal circuits inthe central olfactory nervous system
An astonishing feature of the olfactory system is
its ability to distinguish among more than 400,000
different odorants and among countless number ofodorants-mixtures, each having specific ‘odor’. Using
optical imaging and electrophysiological methods, weare studying the ‘odor maps’ in the central olfactory
system to understand the logic employed by the olfactory system for discrimination among numerous odorants and for perception and learning of the olfactory
image of objects. The target regions for the mapping
include the olfactory bulb and several regions of theolfactory cortex (e.g., piriform cortex and olfactory
tubercle). In addition, odorants having pleasant orunpleasant ‘odor’ are used to map the brain regions
and to elucidate neuronal mechanisms responsible forthe emotional states of the brain.
(2) Neurogenesis and neuron-elimination in theadult brain
Olfactory nervous system has an unusual capacity
to generate neurons throughout life. The sensory neurons in the nasal epithelium and the local interneurons in the olfactory bulb are turning over continuously even in the adult. Therefore, we choose the olfactory system as a model system with which to study themolecular and cellular mechanisms for the recruitment of newly generated neurons into the adult central nervous system and for the selective eliminationof damaged neurons from the functional neuronal circuit. Molecular-genetic and physiological methods are
combined to analyze the mechanisms of the neuronrecruitment and neuron-elimination. The knowledgeof the new-neuron recruitment into the functioning
neuronal circuit should be of critical importance in theremedy of neurological diseases that accompany neuron loss.

36(3) Molecular and cellular mechanisms for theaxonal recognition of specific target neurons andfor the formation of specific neuronal circuits
During development, growing axons, including theaxons from olfactory sensory neurons, can find their
specific target neurons and form functional neuronal
circuits. Recent studies on olfactory sensory neurons
demonstrate a remarkable relationship between theselection of a single odorant receptor gene among arepertoire of 1000 and the address of the axon-projection target-glomeruli in the olfactory bulb. Suchknowledge on the detailed address of the axon targets
is currently available only in the olfactory system. We
apply proteomics approaches to the olfactory axon
address system to elucidate molecular mechanisms
for the olfactory axon guidance to the target glomeruli
and for the formation of specific synaptic connections
with target neurons in the olfactory bulb.
(4) Cellular and molecular mechanisms for thecontact-mediated interactions between neurons
and immune cells in physiological and pathological conditions
Immune cells rarely meet central neurons in physiological conditions because of the blood brain barrier.
However, a massive infiltration of immune cells into
the brain occurs under many pathological conditions,suggesting that immune cells may directly interact
with central neurons. To understand the nature andconsequence of the direct interaction between
immune cells and neurons, we are examining thechange in neuronal morphology and physiologicalstate of primary cultured neurons following the coculture with immune cells. In addition, we are currently focusing on telencephalon-neuron specificmembrane protein, telencephalin (TLCN), which bindto LFA-1 integrin expressed by leukocytes. Since thesoluble form of TLCN can be detected in the serumand cerebrospinal fluid of patients of several neurological diseases, we are developing a sensitive ELISA
assay system to detect the soluble TLCN for monitoring the possible neuronal damage in the telencephalicregions of the patient brain.

Future Prospects
We plan to further pursue the current research
projects that are described above.
Topic (1): In addition to the current studies, wewill start a new project aiming at the elucidation ofneuronal substrate for the olfactory memory. In the
future study an emphasis will be placed on the search
for the behaviorally-relevant olfactory sensory maps
(e.g., food-odor maps) at higher olfactory centers
including the olfactory cortex and amygdala of therodent brain. An emphasis will be placed also on the
search for the neuronal mechanisms associated withodorant-induced emotional changes. These studies
will hopefully provide scientific basis for the therapeutic use of odorants and for the treatment of diseases in the olfactory nervous system.
Topic (2): We pursue to understand molecular andcellular mechanisms how the newly-generated neurons are integrated into the existing neuronal circuitand how the damaged-neurons are removed from thecircuit without damaging the function of the neuronal
circuit in the adult brain.
Topic (3): Based on our initial proteomic approaches, we now have the list of candidate molecules thatmight be involved in the olfactory axon target recognition. Systematic functional assays will be performed
to pin-down the molecular complexes that are responsible for the proper recognition of the olfactory axon
targets. The knowledge of the molecular mechanism
for target recognition in the olfactory system will beof prime importance in understanding the molecularmechanism for the formation of the neuronal circuits
in whole brain regions.
Topic (4): Cellular and molecular analysis of thecontact-mediated interaction between immune cells
and neurons will be further pursued using the co-culture assay system. In addition, we will start a systematic analysis of immune cell-mediated effects in thebrain of the TLCN-deficient mice. Collaborative
research with clinical laboratories will be pursued toestablish the methods for detecting and measuringserum TLCN for the diagnosis of possible damage oftelencephalic neurons in the brain.

Research Grants
1. Grant-in-Aid for Creative Scientific Research (JSPS)(2001-2005, ~65 millions yen / year)
2. Grant-in-Aid for Scientific Research on Priority Areas(B) (MEXT) (1999-2002, ~9.6 millions yen / year)
3. Research Grant from the Human Frontier Science
Program (1999-2001, 59000 USD / year)
4. Grant-in-Aid for Exploratory Research (MEXT)(2001-2002, 1.3 millions yen / year)
5. Research Grant from Mitsubishi Foundation (2000,
7.5 millions yen)

Select Publications (1992~2002)
1. Mori, K, Mataga, N, and Imamura, K. Differential
specificities of single mitral cells in rabbit olfactory
bulb for a homologous series of fatty acid odor molecules. J. Neurophysiol., 67, 786-789, 1992.

2. Kimura, T, Tanizawa, O, Mori, K, Brownstein, M,
and Okayama, H. Structure and expression of ahuman oxytocin receptor. Nature , 356, 526-529,
1992.
3. Tani, A, Yoshihara, Y, and Mori, K. Increase in cytoplasmic free Ca2+ elicited by noradrenalin and serotonin in cultured local interneurons of mouse olfactory bulb. Neuroscience, 49, 193-199, 1992.
4. Imamura, K, Mataga, N, and Mori, K. Coding of odor
molecules by mitral/tufted cells in rabbit olfactory
bulb: I. Aliphatic compounds. J. Neurophysiol., 68,
1986-2002, 1992.
5. Kimura, T, Azuma, C, Saji, F, Takemura, M,
Tokugawa, Y, Miki, M, Ono, M, Mori, K, andTanizawa, O. Estimation by an electrophysiologicalmethod of the expression of oxytocin receptor
mRNA in human myometrium during pregnancy. J.
Steroid Biochem. Mol. Biol., 42, 253-258, 1992.
6. Ishihara, T, Shigemoto, R, Mori, K, Takahashi, K,
and Nagata, S. Functional expression and tissue distribution of a novel receptor for vasoactive intestinal
polypeptide. Neuron, 8, 811-819, 1992.
7. Yoshihara, Y, Katoh, K, and Mori, K. Odor stimulation causes disappearance of R4B12 epitope on axonal surface molecule of olfactory sensory neurons.
Neuroscience, 53, 101-110, 1993.
8. Mochizuki-Oda, N, Negishi, M, Mori, K, and Ito, S.
Arachidonic acid activates cation channels in bovine
adrenal chromaffin cells. J. Neurochem., 61, 18821890, 1993.
9. Mori, K. Molecular and cellular properties of mammalian primary olfactory axons. Microscopy Res.
Technique, 24, 131-141, 1993.
10. Hashimoto, H, Ishihara, T, Shigemoto, R, Mori, K,
and Nagata, S. Molecular cloning and tissue distribution of a receptor for pituitary adenylate cyclase activating polypeptide. Neuron, 11, 333-342, 1993.
11. Katoh, K, Koshimoto, H, Tani, A, and Mori, K.
Coding of odor molecules by mitral/tufted cells in
rabbit olfactory bulb: II Aromatic compounds. J.
Neurophysiol., 70, 2161-2175, 1993.
12. Nemoto, Y, Ikeda, J, Katoh, K, Koshimoto, H,
Yoshihara, Y, and Mori, K. R2D5 antigen: a calciumbinding phosphoprotein predominantly expressed in
olfactory receptor neurons. J. Cell Biol., 123, 963-976,
1993.
13. Mori, K, and Shepherd, G. M. Emerging principles ofmolecular signal processing by mitral/tufted cells in
the olfactory bulb. Seminars in Cell Biol., 5, 65-74,
1994.
14. Koshimoto, H, Katoh, K, Yoshihara, Y, Nemoto, Y,
and Mori, K. Immunohistochemical demonstrationof embryonic expression of an odor receptor protein
and its distribution in the rat olfactory epithelium.
Neurosci. Lett., 169, 73-76, 1994.
15. Yoshihara, Y, Oka, S, Nagata, S, Watanabe, Y,
Kagamiyama, H, and Mori, K. ICAM-related neuronalglycoprotein, telencephalin, with brain segment-specific expression. Neuron, 12, 541-553, 1994.

37

16. Yoshihara, Y, Kawasaki, M, Tani, A, Tamada, A,
Nagata, S, Kagamiyama, H, and Mori, K. BIG-1: a
new TAG-1/F3-related member of the immunoglobulin superfamily with neurite outgrowth-promoting
activity. Neuron, 13, 415-426, 1994.
17. Mori, K, and Yoshihara, Y. Molecular recognition andolfactory processing in the mammalian olfactory system. Prog. Neurobiol., 45, 585-619, 1995.
18. Yokoi, M, Mori, K, and Nakanishi, S. Refinement ofodor molecule tuning by dendrodendritic synaptic
inhibition in the olfactory bulb. Proc. Natl. Acad. Sci.
USA, 92, 3371-3375,1995.
19. Yoshihara, Y, Kawasaki, M, Tamada, A, Nagata, S,
Kagamiyama, H, and Mori, K. Overlapping and differential expression of BIG-2, BIG-1, TAG-1, and F3:
Four members of an AxCAM subgroup of theimmunoglobulin superfamily. J. Neurobiol 28, 51-69,
1995.
20. Mori, K. Relation of chemical structure to specificityof response in olfactory glomeruli. Current Opin.
Neurobiol., 5, 467-474, 1995.
21. Hashimoto, H, Nogi, H, Mori, K, Ohishi, H,
Shigemoto, R, Yamamoto, K, Matsuda, T, Mizuno, N,
Nagata S. and Baba, A. Distribution of the mRNA fora pituitary adenylate cyclase-activating polypeptide
receptor in the rat brain: an in situ hybridization
study. J. Comp. Neurol. 371: 567-577, 1996.
22. Mizuno, T, Yoshihara, Y, Inazawza, Kagamiyama, H.
and Mori, K., cDNA cloning and chromosomal localization of the human telencephalin and its distinctive interaction with LFA-1 integrin. J. Biol. Chem.
272, 1156-1163, 1997.
23. Tian, L, Yoshihara, Y, Mizuno, T, Mori, K, andGahmberg, C G. The neuronal glycoprotein, telencephalin, is a cellular ligand for CD11a/CD18, but not
for CD11b/CD18 and CD11c/CD18 leukocyte integrins. J. Immunol. 158, 928-936, 1997.
24. Fujimori, K E, Takauji, R, Yoshihara, Y, Tamada, A,
Mori, K, and Tamamaki, N. A procedure for in situ
hybridization combined with retrograde labeling of
neurons: Application to the study of cell adhesion
molecule expression in DiI-labeled rat pyramidal
neuron. J. Histochem. and Cytochem. 45, 455-459,
1997.
25. Hino, H, Mori, K, Yoshihara, Y, Iseki, E, Akiyama, H,
Nishimura, T, Ikeda, K and Kosaka K. Reduction oftelencephalin immunoreactivity in the brain ofpatients with Alzheimer’s disease. Brain Res. 753,
353-357, 1997.
26. Yoshihara, Y, Kawasaki, M, Tamada, A, Fujita, H,
Hayashi, H, Kagamiyama, H and Mori, K. OCAM: a
new member of the neural cell adhesion moleculefamily related to zone-to-zone projection of olfactoryand vomeronasal axons. J. Neurosci. 17, 5830-5842,
1997.
27. von Campenhausen, H, Yoshihara, Y, and Mori, K.
OCAM reveals segregated mitral/tufted cell pathways
in developing accessory olfactory bulb. NeuroReport
8, 2607-2612, 1997.

38

28. Yoshihara, Y and Mori, K. Basic principles andmolecular mechanisms of olfactory axon pathfinding. Cell and Tissue Res. 290, 457-463, 1997.
29. Sugino, H, Yoshihara, Y, Copeland, N G, Gilbert, D J,
Jenkins, N A and Mori, K. Genomic organization andchromosomal localization of the mouse telencephalin
gene, a neuronal member of the ICAM family.
Genomics 43, 209-218, 1997.
30. Tamada, A, Yoshihara, Y, and Mori, K. Dendriteassociated adhesion molecule, telencephalin, promotes neurite out growth. Neuroscience Letters, 240,
163-166, 1998.
31. Benson, D, Yoshihara, Y, and Mori, K. Polarized distribution and cell -type specific localization of telencephalin, an ICAM-related neuronal cell adhesion
molecule. J. Neurosci. Res. 52, 43-53, 1998.
32. Sakurai, E, Hashikawa, T, Yoshihara, Y, Kaneko, S,
Sato, M. and Mori, K. Involvement of dendritic adhesion molecule telencephalin in hippocampal longterm potentiation. NeuroReport, 9, 881-886, 1998.
33. Mori, K, Nagao, H. and Sasaki, Y. F. Computation ofmolecular information in mammalian olfactory system. Network: Computation in Neuronal Systems, 9,79-102, 1998.
34. Yoshihara, Y, Mizuno, T, Nakahira, M, Kawasaki, M,
Watanabe, Y, Kagamiyama, H, Jishage, K, Ueda, O,
Suzuki, H, Tabuchi, K, Sawamoto, K, Okano, H,
Noda, T. and Mori, K. A genetic approach to visualization of multi-synaptic neuronal pathways using
plant lectin transgene. Neuron, 22, 33-41, 1999.
35. Arii, N, Mizuguchi, M, Mori, K. and Takashima S.
Development of telencephalin in human cerebrum.
Microscop. Res. Tech. 46, 18-23, 1999.
36. Kashiwadani, H, Sasaki, Y, Uchida, N. and Mori, K.
Synchronized oscillatory discharges of mitral/tufted
cells with different molecular receptive ranges in the
rabbit olfactory bulb. J. Neurophysiol., 82, 17861792, 1999.
37. Mori, K, Nagao. H and Yoshihara, Y The olfactory
bulb: coding and processing of odor molecule information. Science, 286, 711-715, 1999.
38. Mizuno, T, Yoshihara, Y, Kagamiyama, H, Ohsawa,
K, Imai, Y, Kohsaka, S, and Mori, K. Neuronal adhesion molecule telencephalin induces rapid cellspreading of microglia. Brain Research, 849, 58-66,
1999.
39. von Campenhausen, H. and Mori, K. Convergence ofsegregated pheromonal pathways from the accessory
olfactory bulb to the cortex. Eur. J. of Neurosci., 12,33-46, 2000.
40. Tian, L, Kilgannon, P, Yoshihara, Y, Mori, K,
Gallatin,W M, Carpen, O. and Gahmberg, C G.
Binding of T lymphocytes to hippocampal neurons
through ICAM-5 (telencephalin) and characterizationof its interaction with leukocyte integrin
CD11a/CD18. Eur. J. of Immunology, 30, 810-818,
2000.
41. Mori, K, von Campenhausen, H, and Yoshihara, Y.
Zonal organization of the mammalian main andaccessory olfactory systems. Phil. Trans. R. Soc.
Lond. B, 355, 1801-1812, 2000.
42. Yamaguchi, M, Saito, H, Suzuki, M. and Mori, K.
Visualization of neurogenesis in the central nervous
system using nestin promotor-GFP transgenic mice.
NeuroReport, 11, 1991-1996, 2000.
43. Tian, L, Nyman, H, Kilgannon, P, Yoshihara, Y, Mori,K, Anderson, L C, Kaukinen, S, Rauvala, H, Gallatin,
W M and Gahmberg, C G. Intercellular adhesion molecule-5 induces dendritic outgrowth by homophilicadhesion. J. Cell Biol., 150, 243-252, 2000.
44. Nagao, H, Yoshihara, Y, Mitsui, S, Fujisawa, H, andMori, K. Two mirror-image sensory maps withdomain organization in the mouse main olfactorybulb. NeuroReport, 11, 3023-3027, 2000.
45. Uchida, N, Takahashi, Y K, Tanifuji, M, and Mori, K.
Odor maps in the mammalian olfactory bulb: domainorganization and odorant structural features. Nature
Neurosc., 3, 1035-1043, 2000.
46. Nemoto, Y, Kearns, B G, Wenk, M R, Chen, H, Mori,K, Alb, J G Jr., De Camilli, P and Bankaitis, V A
Functional characterization of a mammalian Sac1
and mutants exhibiting substrate-specific defects in
phosphoinositide phosphatase activity. J. Biol.
Chem., 44, 34293-34305, 2000.
47. Arii N, Mizuguchi, M, Mori, K, and Takashima, S.
Ectopic expression of telencephalin in brains withholoprosencephaly. Acta Neuropathol. 100, 506-512,
2000.
48. Tatsura H, Nagao, H, Tamada, A, Sasaki, S, Kohri, K,
Mori, K. Developing germ cells in mouse testis
express pheromone receptors. FEBS letters, 488, 139144, 2001.
49. Mizuno T, Kawasaki, M, Nakahira, M, Kagamiyama,H, Kikuchi, Y, Okamoto, H, Mori, K, and Yoshihara,
Y. Molecular diversity in zebrafish NCAM family:
three members with distinct VASE usage and distinct localization. Mol. and Cell. Neurosci., 18, 119130, 2001.
50. Sawamoto K, Nakao N, Kakishita K, Ogawa Y,
Toyama Y, Yamamoto A, Yamaguchi M, Mori K,
Goldman S A, Itakura T and Okano H. Generation of
dopaminergic neurons in the adult brain from mesencephalic precursor cells labeled with a nestin-GFP
transgene. J. Neurosci., 21, 3895-3903, 2001.
51. Inaki K, Takahashi, Y K, Nagayama, S and Mori K.
Molecular-feature domains with posterrdorsalanteroventral polarity in the symmetrical sensory
maps of the mouse olfactory bulb: mapping ofodourant-induced Zif268 expression. Eur. J. of
Neurosci., 15, 1563-1574, 2002.
52. Sugitani Y, Nakai, S, Minowa, O, Nishi, M, Jishage,
K, Kawano, H, Mori, K, Ogawa, M, and Noda, T. Brn1 and Brn-2 share crucial roles in the production andpositioning of mouse neocortical neurons. Genes andDevelopment, 16, 1760-1765, 2002.
53. Nagao H, Yamaguchi, M, Takahashi, Y, and Mori, K.
Grouping and representation of odorant receptors indomains of the olfactory bulb sensory map.
Microscopy Res. & Technique 58, 168-175, 2002.

39

Department of
Neurophysiology
Outline and Research Objectives
Our laboratory was founded in 1953 as a Department of Brain Physiology in Institute forBrain Research, University of Tokyo Faculty of Medicine, and in 1996 integrated into
University of Tokyo Graduate School of Medicine. We have been teaching Neurophysiology
to undergraduate students in Medical School, and Master and PhD course students. As forresearch, using patch clamp techniques in combination with molecular techniques, we aim
at elucidating cellular and molecular mechanisms underlying synaptic transmission andmodulation. We are particularly interested in dynamic changes at CNS synapses during
postnatal development.
Synapses in the CNS play pivotal roles in neuronal integration and plasticity. During
ontogeny CNS synapses undergo protein reformations, thereby establishing mature and differentiated synaptic functions. Synapses also undergo changes in response to electrical andchemical stimuli. Various proteins in the presynaptic and postsynaptic cells are involved insuch changes, and it is important to determine the role of these proteins in synaptic functions and modulations. To this end, we combine electrophysiological, molecular and morphological techniques to synapses visually identified in the CNS slices. In the rodent auditory brainstem there is a giant excitatory synapse called the calyx of Held. At this synapse it
is possible to make simultaneous whole-cell recordings from a presynaptic terminal and apostsynaptic target cell (Fig. 1). Furthermore, various molecules can be loaded into a nerve
terminal via whole-cell pipette perfusion during the recording (Fig.2). The large structure ofthis synapse also allows us to identify presynaptic proteins immunocytochemically, andeven to follow translocation of presynaptic molecules upon stimulation. With theseapproaches in combination with genetic manipulations and protein overexpressions, we
aim at addressing the molecular basis underlying synaptic functions.

Fig. 1 Simultaneous pre- and postsynaptic wholecell recordings at the calyx of Held
synapse. Inset records on the left show a presynaptic action potential and excitatory postsynaptic
currents (EPSC). Righthand inset illustrates theprocedure of patch-clamp whole-cell recording
method.

Fig. 2 Illustration of pipette perfusion system. A
tube pulled from Eppendorf yellow tip is inserted
into the patch pipette with its tip about 600 mm
from the tip of the patch pipette.

40Faculties and Students
Professor and Chair

Tomoyuki Takahashi MD, PhD(appointed since 1993)
Lecturer
Tetsuhiro Tsujimoto MD, PhD
Associate .................................1
Postdoctral Fellow ..................2
Graduate Students ..................9Technical Assistant ................1
Secretary .................................2

Past Research and Major Accomplishments
1. In simultaneous pre- and postsynaptic recordings
at the calyx of Held synapse, we identified the target of presynaptic inhibition via metabotropic glutamate receptors and GABAB receptors as being
the voltage-dependent Ca2+ channel (Takahashi etal., Science 1996, J Neurosci,1998), and that βγ
subunits of the trimeric G protein Go mediate thepresynaptic inhibition (Kajikawa et al, 2001
PNAS).
2. We identified that phorbol ester stimulates exocytotic machinery downstream of Ca2+ influx thereby
causing synaptic facilitation. By loading inhibitory
peptides into the nerve terminal, we also clarified
that both εPKC (Fig. 4a) and the Munc13-Doc2αinteraction (Fig. 4b) underlie the phorbol esterinduced facilitatory mechanism (Hori et al., J
Neurosci 1998; Saitoh et al, 2002).
3. We found that Ca2+ channel types mediating CNS
synaptic transmission switch during development(Iwasaki et al, J Neurosci 2000).

Fig. 3 Presynaptic inhibitory effect of baclofen occludes that of
L-AP4, suggesting that GABAB and metabotropic glutamate
receptors share a common intracellular mechanism for presynaptic inhibition. Righthand illustration summarizes our current
conclusion. Adenosine A1 receptors also share the same mechanism (Kimura and Takahashi, unpublished observeation).

Fig. 4 Left column, Phorbol etser-induced EPSC potentiation isattenuated by the PKC inhibitor BIS (A) or PKC inhibtitory peptide directly loaded into the calyceal terminal (B). Right column,
The Mid peptide, which interferes with the Doc2α-Munc13-1
interaction attenuated the phorbol etser-induced EPSC potentiation.

4. By loading G protein-related compounds into thecalyx of Held nerve terminal, we clarified that themain role of presynaptic G proteins is to accelerate
the replenishment of synaptic vesicles depleted
after massive release (Takahashi et al. Science2000).
5. We have demonstrated that the quantal analysis
established at the neuromuscular junction is applicable to the CNS synapse (Sahara & Takahashi, J
Physiol 2001).
6. We found that NMDA receptors are unfavorable
for the reliability of fast synaptic transmission,and that their developmental decline regulated byauditory activity at the calyx of Held synapse differentiates the synapse into the high-fidelity one(Futai et al., J Neurosci 2001)

Fig. 5 Synaptic currents mediated by presynaptic N-type Ca2+
channels at 5 different central synapses. At 3 synapses, contribution of N-type Ca2+ channels to synaptic transmission decline
and disappear with postnatal development, whereas at other 2
synapses it remained unchanged throughout development.

7. We found that presynaptic Ca2+ currents undergo
facilitation by repetitive stimulation (Forsythe etal, 1998 Neuron) via acceleration of their gating
kinetics (Cuttle et al., 1998 J Physiol) and that this41

facilitation is mediated by the Ca2+ binding protein
NCS-1 (Tsujimoto et al, 2002 Science).
8. We found that cytoplasmic glutamate concentration in the nerve terminal directly affects thevesicular content of glutamate and that postsynaptic AMPA and NMDA receptors are not saturated
by a single packet of vesicular transmitter(Ishikawa et al, 2002 Neuron).
Fig. 8 Switching the L-glutamate concentration in the presynaptic whole-cell pipette from 1 mM to 100 mM markedly potentiated both quantal (blue) and evoked (red) EPSCs at the calyx ofHeld synapse at physiological temperature.

Current Research

Fig. 6 Postsynaptic action potentials in response to presynaptic
stimulation at 100 Hz at the developing calyx of Held synapse inmice. As mice mature fidelity of transmission increases, whereas blocking NMDA receptors using APV increases the fidelity at
all ages.

1. Identification of presynaptic potassium channels
involved in regulation of transmitter release.
2. Clarification on the developmental changes in thequantal synaptic responses.
3. Mechanism and roles of presynaptic adenosine
receptors.
4. Developmental changes in the size of presynaptic
Ca2+ domain.
5. Identification in the target protein downstream of
εPKC.
6. Clarification in the different properties between
presynaptic N-type and P/Q-type Ca2+ channels.

Future Prospects
1. To elucidate the minimal essential molecules
required for short-term and long-term synaptic
plasticity.
2. To clarify the molecular basis underlying thedevelopmental speeding in the excitatory synaptic
currents.
3. To clarify the presynaptic factor(s) determining
the release probability.

Research Grants (in the past 5 years)
1996-2000
Research for the Future Program by the Japan Societyof Promotion of Sciences “Molecular mechanisms ofcentral synaptic modulation underlying the memory
formation” ¥ 317,092,000
2001-2002
Grant-in-Aid for Specially promoted Research from
the Ministry of Education, Culture, Sports, Scienceand Technology “Cellular and molecular mechanisms
underlying postnatal development and differentiation of central synapses” ¥216,000,000
Fig. 7 The NCS-1 C-terminal peptide, when loaded into thecalyx of Held presynaptic terminal, blocked the activity-dependent facilitations of presynaptic P/Q-type Ca2+ currents in a paired
pulse protocol (A) and in a tetanic stimulation (100 Hz) protocol(C). The peptide had no effect on the current voltage relationship of presynaptic Ca2+ currents.

42

Select Publications
1. Ishikawa T, Sahara, Y, Takahashi T. A single packetof transmitter does not saturate postsynaptic glutamate receptors. Neuron 34, 613-621, 2002.
2. Tsujimoto T, Jeromin A, Saitoh N, Roder JC and
Takahashi T Neuronal calcium sensor 1 and activitydependent facilitation of P/Q-type calcium currents
at presynaptic nerve terminals. Science 275,22762279, 2002.
3. Saitoh N., Hori T. & Takahashi T. Activation of theepsilon isoform of protein kinase C in the mammalian nerve terminal. Proc. Natl. Acad. Sci. USA.
98,14017-14021, 2001.
4. Sahara Y. & Takahashi T. Quantal components of theexcitatory postsynaptic currents at a rat central auditory synapse. J. Physiol. (London) 536,189-197, 2001.
5. Iwasaki S. & Takahashi T. Developmental regulationof transmitter release at the calyx of Held in rat auditory brainstem. J.Physiol. (London) 534,861-871,
2001.
6. Kajikawa Y., Saitoh N.& Takahashi T. GTP-binding
protein βγ subunits mediate presynaptic calcium current inhibition by GABAB receptor. Proc. Natl. Acad.
Sci.U.S.A 98,8054-8058, 2001.
7. Ishikawa T. & Takahashi T. Mechanisms underlying
presynaptic facilitatory effect of cyclothiazide at thecalyx of Held of juvenile rats. J. Physiol. (London)
533, 423-431, 2001.
8. Futai K., Okada M., Matsuyama K. & Takahashi T.
High-fidelity transmission acquired via a developmental decrease in NMDA receptor expression at anauditory synapse. J. Neurosci. 21,3342-3349, 2001.
9. Yamauchi, T., Hori T. & Takahashi T. Presynaptic
inhibition by muscimol through GABAB receptors.
Eur. J. Neurosci. 12, 3433-3436, 2000.
10. Takahashi T., Hori T., Kajikawa Y.& Tsujimoto T.
The role of GTP-binding protein activity in fast central synaptic transmission. Science 289, 460-463,
2000.
11. Okada M., Onodera K., Van Renterghem C., Sieghart
W.& Takahashi T. Functional correlation of GABAA
receptor α subunits expression with the properties
of IPSCs in the developing thalamus. J. Neurosci. 20,
2202-2208, 2000.
12. Iwasaki S., Momiyama A., Uchitel O.D.& Takahashi
T. Developmental changes in calcium channel types
mediating central synaptic transmission. J. Neurosci.
20, 59-65, 2000.
13. Hori T., Takai Y. & Takahashi T. Presynaptic mechanism for phorbol ester- induced synaptic potentiation. J. Neurosci. 19, 7262-7267, 1999.
14. Terada S., Tsujimoto T., Takei Y., Takahashi T. &Hirokawa N. Impairement of inhibitory synaptictransmission in mice lacking synapsin 1. J. Cell Biol.
145, 1039-1048, 1999.
15. Kobayashi K., Manabe T. & Takahashi T. Calciumdependent mechanisms involved in presynaptic
long-term depression at the hippocampal mossy

fibre-CA3 synapse. Eur. J. Neurosci. 11, 1633-1638,
1999.
16. Mori H., Manabe T., Watanabe M., Satoh Y., SuzukiN., Toki S., Nakamura K., Yagi T., Kushiya E.,
Takahashi T., Inoue Y., Sakimura K. & Mishina M.
Role of the carboxyl-terminal region of the GluR ε2subunit in synaptic localization of the NMDA receptor channel. Neuron 21, 571-580, 1998.
17. Manabe T., Noda Y., Mamiya T., Katagiri H., HoutaniT., Nishi M., Noda T., Takahashi T., Sugimoto T.,
Nabeshima T. & Takeshima H. Facilitation of longterm potentiation and memory in mice lacking nociceptin receptors. Nature 394, 577-581, 1998.
18. Cuttle M. F, Tsujimoto T., Forsythe I.D. & Takahashi
T. Facilitation of the presynaptic calcium current at
an auditory synapse in rat brainstem. J.
Physiol.(London) 512, 723-729, 1998.
19. Iwasaki S. & Takahashi T. Developmental changes incalcium channel types mediating synaptic transmission in rat auditory brainstem. J. Physiol.(London)
509, 419-423, 1998.
20. Takahashi T., Kajikawa Y. & Tsujimoto T. G-proteincoupled modulation of presynaptic calcium currents
and transmitter release by a GABA B receptor. J.
Neurosci. 18, 3138-3146, 1998.
21. Forsythe I.D., Tsujimoto T., Barnes-Davies M., Cuttle
M.F., & Takahashi T. Inactivation of presynaptic calcium current contribute to synaptic depression at afast central synapse. Neuron 20, 797-807, 1998.
22. Takahashi T., Forsythe I., Tsujimoto T., BarnesDavies M. & Onodera K. Presynaptic calcium current
modulation by a metabotropic glutamate receptor.
Science 274, 594- 597, 1996.
23. Kobayashi K., Manabe T. & Takahashi T. Presynaptic
long-term depression at the hippocampal mossy
fiber-CA3 synapse. Science 273, 648-650, 1996.
24. Yokoi M., Kobayashi K., Manabe T., Takahashi T.,
Sakaguchi I., Katsuura., Shigemoto R., Ohishi H.,
Nomura S., Nakamura K., Nakao K., Katsuki M., &Nakanishi S. Impairment of hippocampal mossy
fiber LTD in mice lacking metabotropic glutamate
receptor subtype 2. Science 273, 645-647, 1996.
25. Takahashi T., Feldmeyer D., Suzuki N., Onodera K.,
Cull-Candy S.G., Sakimura K., & Mishina M.
Functional Correlation of NMDA receptor ε subunits
expression with the properties of single-channel andsynaptic current in the developing cerebellum. J.
Neurosci. 16, 4376-4382, 1996.
26. Silver R.A., Cull-Candy S.G. & Takahashi T. NonNMDA glutamate receptor occupancy and open
probability at a rat cerebellar synapse with single
and multiple release sites. J. Physiol. (London) 494,
231-250, 1996.
27. Kutsuwada T., Sakimura K., Manabe T., Takayama
C., Katakura N., Kushiya E., Natsume R., Watanabe
M., Inoue Y., Yagi T., Aizawa S., Arakawa M.,
Takahashi T., Nakamura Y., Mori H. & Mishina M.
Impairment of suckling response, trigeminal neuronal pattern formation and hippocampal LTD in

43

NMDA receptor ε2 subunit mutant mice. Neuron 16,
333-344, 1996.
28. Farrant M., Feldmeyer D., Takahashi T. & CullCandy S.G. NMDA-receptor channel diversity in thedeveloping cerebellum. Nature 368, 335-339, 1994.
29. Momiyama A. & Takahashi T. Calcium channels
responsible for potassium-induced transmitter
release at rat cerebellar synapses. J Physiol. (London)
476, 197-202, 1994.
30. Takahashi T. & Momiyama A. Different types of calcium channels mediate central synaptic transmission. Nature 366,156-158, 1993.

44

Department of Cellular andMolecular Pharmacology
Outline and Research Objectives
Our department was founded in 1885 as the first pharmacology department in Japan. It
has had a strong research background in the field of calcium (Ca2+) signalling since Professor
Emeritus Setsuro Eabashi discovered in 1950’s the regulatory role of intracellular Ca2+ concentration in muscle contraction. Since then the field of Ca2+ signalling research has expanded extensively: the Ca2+ signal is now known as a molecular switch in a vast array of important cell functions including muscle contraction, exocytosis, cell proliferation, immune
responses and regulation of synaptic functions. The present research group led by Professor
Iino is interested in the general principle of the Ca2+ signalling mechanism and is particularlyinterested in Ca2+ signalling in neurons and muscle cells at present. We have recently
expanded the scope of our research to various signaling molecules upstream and downstream of Ca2+ signals. A notable feature of our department is that we have had an assemblyof staff members with diverse backgrounds, e.g., cell physiology, molecular biology and neurobiology. We believe that the collaboration of these people will facilitate the elucidation of
the medical and biological significance of Ca2+ signalling and related subjects.

Faculties and Students
Professor

Masamitsu Iino, M.D., Ph.D.
(From April 1995)
Associate Professor Kenzo Hirose, M.D., Ph.D.
Associate .................................2
Postdoctoral Fellows...............3
Graduate Students ................11
Research Students...................3
Laboratory Staff ......................3

Past Research and Major Accomplishments
Our research focuses on the regulation of Ca2+ signals. In particular, we have made important contributions to clarifying intracellular Ca2+ release mechanisms. Our research also includes signaling molecules
upstream and downstream of Ca2+ signals. Throughthese works, we have clarified feedback mechanismsthat are essential to the generation of spatiotemporal
patterns of Ca2+ signals.

1) Functional properties of IP3R.
The receptor-mediated activation of phospholipase
C results in the production of inositol 1,4,5-trisphosphate (IP3), which then releases Ca2+ from the intracellular Ca2+ store. This Ca2+ mobilization is a central
mechanism in the generation of Ca2+ signals for the
regulation of various cell functions. We showed forthe first time that the Ca2+ release via IP3R is activatedby submicromolar concentrations of Ca2+ (Fig. 1). We
also discovered an inhibitory role of Ca2+ at higher
concentrations. Thus, IP 3R activity is biphasicallydependent on cytosolic Ca2+ concentration with the
maximum level of activation obtained at 0.3 µM (J.
Gen. Physiol., 1990). We also showed that the effectsof Ca2+ on IP3R activity have no notable delay in experiments using both caged IP3 and caged Ca2+ (Nature,1992). These results suggest the presence of a positive
feedback loop in IP3R-mediated Ca2+ release. Such a
positive feedback loop is expected to have a major
effect on Ca2+ release kinetics via IP3R (Nature, 1994b).
This notion, which is very popular in the field of Ca2+
signalling, now has a strong molecular basis as shown
below.
We then studied the structure-function relationship of IP3R. IP3R consists of three subtypes (IP3R1,
IP3R2 and IP3R3), that are encoded by different genes
and are expressed in a tissue- and development-specific manner. In collaboration with Prof. Kurosaki of
Kansai Medical University, we genetically engineered
DT40 B cells to express only one IP3R subtype andFigure 1. IP3 and Ca2+ dependence of IP3R activity. Both IP3 andCa2+ are required in the activation of IP3R.

45

showed that each IP3R subtype has distinct properties(EMBO J., 1999). For example, the order of IP3 sensitivity is IP3R2 > IP3R1 >I P3R3. Furthermore, we succeeded in mapping the Ca2+ sensor region of IP3R1 at
glutamate 2100 (Fig. 2) (EMBO J., 2001). Substitutionof this amino acid by aspartate (E2100D) resulted in a
10-fold decrease in the Ca2+ sensitivity of IP3R1. When
we expressed the E2100D mutant IP3R in DT40 cells,
the rates of increase of Ca2+ concentration and Ca2+
oscillations during an agonist-induced response weresignificantly reduced compared with cells expressing
wild-type IP3R1 (Fig. 3). These results demonstratethat the Ca2+-mediated feedback regulation of IP3R
activity is extremely important for the generation of

Figure 2. Ca2+ sensor of IP3R. Glutamate (E) at position 2100
functions as the Ca2+ sensor of IP3R.

Figure 3. Ca2+ signalling in cells expressing mutant (E2100D)
IP3R with reduced Ca2+ sensitivity. BCR-mediated Ca2+ oscillation was abolished in cells expressing the mutant IP3R.

Figure 4. Positive feedback regulation of IP3R via the Ca2+ sensor. Ca2+-sensor-mediated positive feedback regulation of Ca2+
release via IP3R is essential for the spatio-temporal pattern generation of agonist-stimulated Ca2+ release.

46

spatiotemporal patterns of Ca2+ signals (Fig. 4).

2) Ca2+ imaging using intact tissues.
In order to fully understand the physiological functions of Ca2+-mediated cell signalling mechanisms, it isimportant to study Ca2+ signalling in cells that communicate with each other within intact tissues. With
this notion in mind, we performed Ca 2+ imaging
experiments using intact vascular tissues. Thismethod successfully revealed that vascular smoothmuscle cells respond to sympathetic nerve stimuli
with Ca2+ waves and oscillations (EMBO J., 1994). To
our surprise, resting arterial smooth muscle cells were
not quiescent in terms of Ca2+ signals. We discovered
spontaneous Ca2+ transients in unstimulated vascularsmooth muscle cells (J. Physiol., 1999). These Ca2+transients are called “Ca2+ ripples” because their peak
amplitudes were much smaller than those of sympathetic nerve-stimulated Ca2+ responses. Ca2+ ripples
contribute to some extent to the resting tonus of thevascular wall. Other physiological functions await further clarification.
We have now extended the application of the tissue imaging method to intestinal tissues, and have forthe first time succeeded in simultaneous imaging of
Ca2+ signals in smooth muscle cells and interstitialcells of Cajal, which are the putative intestinal pacemaker cells (J. Physiol., 2002). This method may help
us elucidate the rhythm-making mechanism in intestinal tissues.
3) Excitation-contraction coupling and malignant
hyperthermia.
In skeletal muscle cells depolarization of the plasma membrane induces Ca2+ release via the Ca2+ releasechannel (ryanodine receptor, RyR) on the adjacent sarcoplasmic reticulum. This process is called excitationcontraction (EC) coupling and has been studied using
various methods. We have made several important
contributions to clarifying the molecular basis of E-C
coupling. There are three subtypes of RyR (RyR1,
RyR2 and RyR3) encoded by different genes. First, we
showed that RyR1 is critically important for EC coupling (Nature, 1994a). Second, we showed that RyR1
but not RyR2 or RyR3 supports the skeletal muscle EC
coupling (EMBO J., 1996). Third, a stretch of about
100 amino acids (D2 region), which is ~1,300 aminoacids removed from the N terminus of RyR1, is important for EC coupling ( J. Biol. Chem. , 1997). These
results provided a framework for the molecular elucidation of EC coupling.
In relation to this subject, we have identified oneof the genetic causes of malignant hyperthermia, acomplication of general anesthesia with a life-threatening fever, resulting from an abnormality in theskeletal muscle calcium release channel, RyR1. We

identified a point mutation (L4838V) that is responsible for the gain-of-function mutation of RyR1 in
malignant hyperthermia patients with marked clinicalsymptoms (Jpn. J. Pharmacol., 2002).
We also discovered junctophilin, a protein that is
responsible for the junction formation between theplasma membrane and the sarcoplasmic reticulum
membrane (Mol. Cell, 2000).

4) IP3 imaging and discovery of a new IP3 signallingmechanism.
We have succeeded in visualizing the changes inintracellular IP3 concentration using the translocationof the GFP-tagged PH domain of PLC-δ1 (GFP-PHD)(Science, 1999). Using this method, we showed for the
first time the dynamic changes in the intracellular IP3
concentration (IP3 oscillations and waves) (Fig. 5).
These results show that the IP3 dynamics is an important factor for the generation of dynamic spatiotemporal patterns of Ca2+ signals. Furthermore, we applied
this method to measuring the changes in IP3 concentration in cerebellar Purkinje cells and found a novel
Ca2+-mediated IP3 signalling pathway that leads to IP3
production following climbing fiber inputs into
Purkinje cells (Neuron, 2001) (Fig. 6).

Current Research
Based on the results of our studies of Ca 2+ signalling, we recognize the importance of the spatiotemporal distribution of signalling molecules in defining
cell signals. Thus, global Ca2+ waves and oscillations insmooth muscle cells result in contraction of the cells,
while localized, transient rises at subplasmalemmal
regions (Ca2+ sparks) result in relaxation of the cells.
We, therefore, believe that it is extremely importantto visualize the spatiotemporal distribution of signalling molecules within intact cells. To that end, weare currently engaged in the development of indicators of important signalling molecules (see below).
Two types of excitable cells, neurons of the central
nervous system and smooth muscle cells, are our current target cells. In particular, neurons have distinct
cell polarity, therefore the spatiotemporal distributionof signalling molecules should be of great importancefor the functions of neurons.

1) Development of novel signal indicators.
We are currently involved in the development of
new genetically coded indicators of various importantcell signals including nitric oxide (NO), protein phosphorylation and a Ca2+-dependent transcription factor.
Some of the indicators are now being expressed incells for analysis, and others are now close to application to cells.

2) Ca2+ signalling in Purkinje cells and synaptic
plasticity.
Ca2+ and IP3 dynamics in Purkinje cells are now
studied in Purkinje cells in conjunction with themolecular basis of synaptic plasticity. We use bothconfocal and two-photon excitation microscopies toobserve signals in individual dendrites.
3) Molecular approaches to the study of IP3Rmediated Ca2+ signalling.
We have identified the Ca2+ sensor region of IP3R
and showed that a mutation at this site resulted in
the inhibition of Ca2+ signals. We are introducing thismutation to various cells to inhibit Ca 2+ signals.
Through this approach it will be possible to clarify theroles of Ca2+ signals in various cells.

Future Prospects
1) Elucidation of the relationship between Ca2+
signalling and cell functions.
Intracellular Ca2+ signals exhibit extremely dynamic changes both temporally and spatially. Such property allows the Ca2+ signal to be an extremely versatileswitch regulating diverse cell functions; from transmitter release in neurons to cell proliferation, andfrom muscle contraction to apoptosis. The role of Ca2+
signals in skeletal muscle contraction has been thoroughly clarified. However, there are still many cellfunctions in which Ca2+ signals are thought to play
important regulatory roles, but their mechanisms
remain elusive. We would like to elucidate the relationship between the Ca2+ signalling mechanism andthe regulation of cell functions in all such frontiers.
2) Visualization and analysis of molecular events
at synapses during synaptic plasticity.
Among many cell functions, we are particularlyinterested in those of neurons, which have such aunique cell polarity and provide an excellent platform
for spatiotemporal cell signalling. We are interested incell signals upstream and downstream of the Ca2+ signals. Combining new signal indicators and imaging
methods, we wish to visualize the spatiotemporal distribution of cell signals in order to elucidate molecular
events at the synapses during synaptic plasticity that
underlies learning and memory.

Research Grants
1. Core Research for Evolutional Science andTechnology (CREST) from Japan Science andTechnology Cooperation “Calcium SignallingResearch with Advancement of Imaging and47

Molecular Genetic Methods“ (Years 1996-2000)¥676,312,000.
2. Grant-in-Aid for Scientific Research (Specially
Promoted Research) from the Minisitry of Education,
Science, Sports and Culture. “Visualization of
Intracellular Signal Flow” (Years 2000-2004)¥430,000,000.

Select Publications (Reprints of the ten references with asterisks are attached.)
1. Iino, M. Tension responses of chemically skinned
fibre bundles of the guinea-pig taenia caeci undervaried ionic environments. J. Physiol. 320, 449-467,
1981.
2. Iino, M. Calcium dependent inositol trisphosphateinduced calcium release in the guinea-pig taenia
caeci. Biochem. Biophys. Res. Commun. 142, 47-52,
1987.
3. Iino, M. Calcium induced calcium release mechanism
in guinea-pig taenia caeci. J. Gen. Physiol. 94, 363383, 1989.
4.* Iino, M. Biphasic Ca2+ dependence of inositol 1,4,5trisphosphate-induced Ca release in smooth muscle
cells of the guinea pig taenia caeci. J. Gen. Physiol.
95, 1103-1122, 1990.
5. Iino, M. Effects of adenine nucleotides on inositol
1,4,5-trisphosphate-induced calcium release in vascular smooth muscle cells. J. Gen. Physiol. 98, 681-698,
1991.
6.* Iino, M. and Endo, M. Calcium-dependent immediate
feedback control of inositol 1,4,5-trisphosphateinduced Ca2+ release. Nature 360, 76-78, 1992.
7. Oyamada, H., Iino, M. and Endo, M. Effects of ryanodine on the properties of Ca2+ release from the sarcoplasmic reticulum in skinned skeletal muscle fibresof the frog. J. Physiol. 470, 335-3348, 1993.
8. Iino, M., Yamazawa, T., Miyashita, Y., Endo, M. andKasai, H. Critical intracellular Ca2+ concentration for
all-or-none Ca 2+ spiking in single smooth muscle
cells. EMBO J. 12, 5287-5291., 1993
9. Tsukioka, M., Iino, M. and Endo, M. pH dependence
of inositol 1,4,5-trisphosphate-induced Ca2+ release inpermeabilized smooth muscle cells of the guinea-pig.
J. Physiol. 475, 369-375, 1994.
10. Oyamada, H., Murayama, T., Takagi, T., Iino, M.,
Iwabe, N., Miyata, T. Ogawa, Y. and Endo, M.
Primary structure and distribution of ryanodinebinding protein isoforms of the bullfrog skeletal
muscle. J. Biol. Chem. 269, 17206-17214, 1994.
11. Kasai, Y., Iino, M., Tsutsumi, O., Taketani, Y. andEndo, M. Effects of cyclopiazonic acid on rhythmic
contractions in uterine smooth muscle bundles of
the rat. Br. J. Pharmacol. 112, 1132-11136, 1994.
12.* Takeshima, H., Iino, M., Takekura, H., Nishi, M.,
Kuno, J., Minowa, O., Takano, H. and Noda, T.
Excitation-contraction uncoupling and musculardegeneration in mice lacking functional skeletal muscle ryanodine-receptor gene. Nature 369, 556-559,
1994.

48

13.* Iino, M., Kasai, H. and Yamazawa, T. Visualizationof neural control of intracellular Ca2+ concentration insingle vascular smooth muscle cells in situ. EMBO J.
13, 5026-5031, 1994.
14.* Hirose, K. and Iino, M. Heterogeneity of channel
density in inositol-1,4,5-trisphosphate- sensitive Ca2+
stores. Nature 372, 791–794, 1994.
15. Kasai, Y., Tsutsumi, O., Taketani, Y., Endo, M. andIino, M. Stretch-induced enhancement of contractions in uterine smooth muscle of rats. J. Physiol.
486, 373-384, 1995
16. Takeshima, H., Yamazawa, T., Ikemoto, T.,
Takekura, H., Nishi, M., Noda, T. and Iino, M. Ca2+induced Ca2+ release in myocytes from dyspedic micelacking type-1 ryanodine receptor. EMBO J. 14,
2999–3006, 1995.
17. Ikemoto, T., Iino, M. and Endo, M. Enhancing effectof calmodulin on Ca2+-induced Ca2+ release in the sarcoplasmic reticulum of rabbit skeletal muscle fibres.
J. Physiol. 487, 573-582, 1995.
18. Ikemoto, T., Iino, M. and Endo, M. Effect of calmodulin antagonists on calmodulin-induced biphasic
modulation of Ca 2+ -induced Ca 2+ release. Br. J.
Pharmacol. 118, 690-694, 1996.
19. Takeshima, H., Ikemoto, T., Nishi, M., Nishiyama,N., Shimuta, M., Sugitani, Y., Kuno, J., Saito, I., Saito,
H., Endo, M., Iino, M. and Noda, M. Generation andcharacterization of mutant mice lacking ryanodine
receptor type 3. J. Biol. Chem. 271, 19649-19652,
1996.
20.* Yamazawa, T., Takeshima, H., Sakurai, T., Endo, M.
and Iino, M. Subtype specificity of ryanodine receptor for Ca2+ signal amplification in excitation-contraction coupling. EMBO J. 15, 6172-6177, 1996.
21. Yamazawa,T., Takeshima, H., Shimuta, M. and Iino,
M. A region of the ryanodine receptor critical for
excitation-contraction coupling in skeletal muscle. J.
Biol. Chem. 272, 8161-8164, 1997.
22. Ikemoto, T., Komazaki, S., Takeshima, H., Nishi, M.,
Noda, T., Iino, M. and Endo, M. Functional and morphological features of skeletal muscle from mutant
mice lacking both type 1 and type 3 ryanodine receptors. J. Physiol. 501, 305-312, 1997.
23. Kasai, Y., Yamazawa, T., Sakurai, T., Taketani, Y.
and Iino, M. Endothelium-dependent frequency
modulation of Ca2+ signaling in individual vascularsmooth muscle cells of the rat. J. Physiol. 504, 349357, 1997.
24. Takesako, K., Sasamoto, K., Ohkura, Y., Hirose, K.
and Iino, M. Low-affinity fluorescent indicator for
intracellular calcium ions. Anal. Commun. 34, 391392, 1997.
25. Hirose, K., Kadowaki, S. and Iino, M. Allosteric regulation by cytoplasmic Ca2+ and IP3 of the gating of IP3
receptors in guinea pig vascular smooth muscle cells.
J. Physiol. 506, 407-414, 1998.
26. Takeshima, H., Komazaki, S., Hirose, K., Nishi, M.,
Noda, T. and Iino, M. Embryonic lethality andabnormal cardiac myocytes in mice lacking ryanodine receptor type 2. EMBO J. 17, 3309-3316, 1998.

27. Takeshima, H., Shimuta, M., Komazaki, S., Ohmi, K.,
Nishi, M., Iino, M., Miyata, A. and Kangawa, K.
Mitsugumin29, a novel synaptophysin family member from the triad junction in skeletal muscle.
Biochem. J. 331, 317-322, 1998.
28. Okada, H., Bolland, S., Hashimoto, A., Kurosaki, M.,
Kabuyama, Y., Iino, M., Ravech, J.V., Kurosaki, T.
Role of the inositol phosphatase SHIP in B cell receptor-induced Ca2+ oscillatory response. J. Immunol.
161, 5129-5132, 1998.
29. Ikemoto, T., Takeshima, H. Iino, M. and Endo, M.
Effect of calmodulin on Ca2+-induced Ca2+ release ofskeletal muscle from mutant mice expressing either
ryanodine receptor type 1 or type 3. Pflügers Arch.
437, 43-48, 1998.
30. Manabe, T., Noda, Y., Mamiya, T., Katagiri, H.,
Houtani, T., Nishi, M., Noda, T., Takahashi, T.,
Sugimoto, T., Nabeshima, T. and Takeshima, H.
Facilitation of long-term potentiation and memory inmice lacking nociceptin receptor. Nature 394, 577581, 1998.
31. Hirabayashi, T., Kume, K., Hirose, K., Yokomizo, T.,
Iino, M., Itoh, H. and Shimizu, T. Critical duration of
intracellular Ca2+ response required for continuoustranslocation and activation of cytosolic phospholipase A2. J. Biol. Chem. 274, 5163-5169, 1999.
32.* Miyakawa, T., Maeda, A., Yamazawa, T., Hirose, K.,
Kurosaki, T. and Iino, M. Encoding of Ca2+ signals bydifferential expression of IP 3 receptor subtypes.
EMBO J. 18, 1303-1308, 1999.
33. Hashimoto, A., Hirose, K., Okada, H., Kurosaki, T.
and Iino, M. Inhibitory modulation of B cell receptormediated Ca2+ mobilization by SH2 domain containing inositol 5’-phosphatase (SHIP). J. Biol. Chem. 274,
11203-11208, 1999.
34. Hirose, K., Takeshima, H. and Iino, M. Fluorescentindicators for inositol 1,4,5-triphosphate based on
bioconjugates of pleckstrin homology domain andfluorescent dyes. Anal. Commun. 36, 175-177, 1999.
35.* Hirose, K., Kadowaki, S., Tanabe, M., Takeshima,
H. and Iino, M. Spatio-temporal dynamics of Inositol
1,4,5-trisphosphate that underlies complex Ca 2+
mobilization patterns. Science 284, 1527-1530, 1999
36. Asada, Y, Yamazawa, T., Hirose, K., Takasaka, T.
and Iino, M. Dynamic signaling in rat arterial smoothmuscle cells under the control of local reninangiotensin system. J. Physiol. 521, 497-505, 1999.
37. Nishi, M. Komazaki, S., X Kurebayashi, N., Ogawa,Y., Noda, T., Iino, M. and Takeshima, H. Abnormalfeatures in skeletal muscle from mice lacking
Mitsugumin29. J. Cell Biol. 147, 1473-1480, 1999
38. Takeshima, H., Komazaki, S., Nishi, M., Iino, M. andKangawa, K. Junctophilins: a novel family of junctional membrane complex proteins. Molecular Cell,6, 11-22, 2000.
39. Hashimoto, A., Hirose, K., Kurosaki, T. and Iino, M.
Negative control of store-operated Ca2+ influx by B
cell receptor crosslinking. J. Immunol. 166, 10031008, 2001.

40. Nemoto, T., Kimura, R., Ito, K., Tachikawa, A.,
Miyashita, Y., Iino, M. and Kasai, H. Sequentialreplenishment mechanism of exocytosis in pancreatic acini. Nature Cell Biol. 3, 253-258, 2001.
41. Inoue, T., Kikuchi, K., Hirose, K., Iino, M. andNagano, T. Small molecule-based laser inactivation of
inositol 1,4,5-trisphosphate receptor. Chem. Biol. 8,9-15, 2001.
42.* Miyakawa, T., Mizushima, A., Hirose, K.,
Yamazawa, T., Bezprozvanny, I., Kurosaki, T. andIino, M. Ca2+-sensor region of IP3 receptor controls
intracellular Ca2+ signalling. EMBO J. 20, 1674-1680,
2001.
43. Fujiwara, A., Hirose, K., Yamazawa, T. and Iino, M.
Reduced IP3 sensitivity of IP3 receptor in Purkinje
neurons. NeuroReport 12, 2647-2651 2001.
44.* Okubo, Y., Kakizawa, S., Hirose, K. and Iino, M.
Visualization of IP3 dynamics reveals a novel AMPA
receptor-triggered IP3 production pathway mediatedby voltage-dependent Ca2+ influx in Purkinje cells.
Neuron 32, 113-122, 2001.
45. Namikis, S., Hirose, K. and Iino, M. Mapping ofheme-binding domains in soluble guanylyl cyclase
β1 subunit. Biochem. Biophys. Res. Commun. 288,
798-804, 2001.
46. Matsuzaki, M., Ellis-Davies, G.C.R., Nemoto, T.,
Miyashita, Y., Iino, M. and Kasai, H. Dendritic spine
geometry is critical for AMPA receptor expression in
hippocampal CA1 pyramidal neurons. Nature
Neurosci. 4, 1086-1092, 2001.
47. Yamazawa, T. and Iino, M. Simultaneous imaging of
Ca2+ signals in interstitial cells of Cajal and longitudinal smooth muscle cells during rhythmic activity inmouse ileum. J. Physiol. 538, 823 –835, 2002.
48. Oyamada, H., Oguchi, K., Saitoh, N., Yamazawa, T.,
Hirose, K., Kawana, Y., Wakatsuki, K., Oguchi, K.,
Tagami, M., Hanaoka, K., Endo, M. and Iino, M.
Novel mutations in C-terminal channel region of theryanodine receptor in malignant hyperthermia
patients. Jpn. J. Pharmacol. 88, 159-166, 2002.
49. Tu, H., Miyakawa, T., Wang, Z., Glouchankova, L.,
Iino, M. and Bezprozvanny, I. Functional characterization of the type 1 Inositol 1,4,5-trisphosphate
receptor coupling domain SII({+/-}) splice variants
and the opisthotonos mutant form. Biophys. J. 82,
1995-2004, 2002.
50. Ueda, H.R., Chen, W., Adachi, A., Wakamatsu, H.,
Hayashi, S., Takasugi, T., Nagano, M., Nakahama,
K.-I., Suzuki, Y., Sugano, S., Iino, M., Shigeyoshi, Y.
and Hashimoto, S. A transcription factor response
element for gene expression during circadian night.
Nature, 418, 534 – 539, 2002.

49

Department of
Molecular Neurobiology
Outline and Research Objective
We have been investigating the molecular mechanism of brain functions. Current
research activities are focused on the glutamate receptor (GluR) and learning and memory.
We elucidated the molecular diversity of the N-methyl-D-aspartate (NMDA)-type GluR anddiscovered the novel δ subfamily of GluR by molecular cloning. Roles of these GluRs inbrain functions have been studied by gene targeting.

Faculties and Students
Professor and Chair Masayoshi Mishina, Ph.D (1994~)
Lecturer
Hisashi Mori, Ph.D
Associate .................................2 (Naoto Matsuda, M.D andTomonori Takeuchi, Ph.D)
Postdoctoral Fellow ................2
Graduate Student..................11
Research Student ....................2
Secretary .................................2

Past Research and Major Accomplishments
The NMDA subtype of GluR is unique in functional properties among many neurotransmitter receptors
and ion channels mediating neural signaling in thebrain. The NMDA receptor channel is gated both byligands and by voltage, and is highly permeable toCa2+. These characteristics of the NMDA receptor
directly relate to its important physiological roles insynaptic plasticity as a molecular coincidence detector. Some forms of long-term potentiation (LTP) andlong-term depression (LTD), which are thought tounderlie learning and memory, are critically dependent on the NMDA receptor channel.

Molecular characterization of the NMDA receptor
We elucidated the molecular diversity of the
NMDA receptor by molecular cloning. Highly active
NMDA receptor channel was formed in vitro by coexpression of two members of GluR subunit families,that is, the GluRε (NR2) and GluRζ (NR1). There are
four members in the ε subunit family, whereas only
one member is known in the GluRζ subunit family
except for the splice variants. All of the NMDA receptor channel subunits possess asparagine in segment
M2. Replacement by glutamine of the asparagine insegment M2 of the GluRε2 and GluRζ1 strongly
reduced the sensitivity to Mg2+ block of the NMDA
receptor channel. Since there is strong evidence that
Mg2+ produces a voltage-dependent block of the channel by binding a site deep within the ionophore, these50

results are consistent with the view that segment M2
constitutes the ion channel pore of the NMDA receptor channel.
At the embryonic stages, the GluRε2 (NR2B) subunit mRNA is expressed in the entire brain, and the
GluRε4 (NR2D) subunit mRNA in the diencephalon
and the brainstem. After birth, the GluRε1 (NR2A)
subunit mRNA appears in the entire brain, and the
GluRε3 (NR2C) subunit mRNA mainly in the cerebellum. The expression of the GluRε2 subunit mRNA
becomes restricted to the forebrain, and that of the
GluRε4 subunit mRNA is strongly reduced. The
GluRζ1 subunit mRNA is found ubiquitously in thebrain during development. The four GluRε subunits
are also distinct in functional properties and regulation. Thus, multiple GluRε subunits are major determinants of the NMDA receptor channel diversity, andthe molecular compositions and functional properties
of NMDA receptor channels are different dependingon the brain regions and developmental stages. Thesefindings raise an important question whether themolecular diversity underlies the various physiologicalroles of the NMDA receptor channel.

Physiological roles of multiple NMDA receptor
subtypes
To examine the functional roles in vivo of thediverse NMDA receptor subtypes, we generated
mutant mice defective in respective GluRε subunits bygene targeting. Disruption of the GluRε1 gene results
in reduction of hippocampal LTP and impairment of
Morris water maze learning. In GluRε1 mutant mice,
thresholds for both hippocampal LTP and contextual
learning increased. The ablation of the GluRε2 subunit also impaired synaptic plasticity in the hippocampus. The reduction of GluRε1 and GluRε2 affected theplasticity of the hippocampal CA3 region in a synapsespecific manner. These results indicate that the
NMDA receptor GluRε1 and GluRε2 subtypes play a
key role in synaptic plasticity, learning and memory.
GluRε2 mutant mice died shortly after birth andfailed to form the whisker-related neural pattern (bar-relettes) in the brainstem trigeminal complex. In contrast, the barrelette formation was normal in GluRε4mutant mice. These results show the involvement of
the GluRε2 subunit in the refinement of the synapse
formation of periphery-related neural patterns in the
mammalian brain. Heterozygous mutant mice with
reduced GluRε2 subunit of the NMDA receptor
showed strongly enhanced startle responses toacoustic stimuli. On the other hand, heterozygous andhomozygous mutation of the other NMDA receptor
GluRε subunits exerted little or only small effects on
acoustic startle responses. Thus, the NMDA receptor
GluRε2 plays a role in the regulation of the startle
reflex. GluRε4 mutant mice exhibited reduced spontaneous activity, while GluRε3 mutant mice showed little obvious deficit. GluRε1 and GluRε4 differentiallycontributed to pain modulation.

Discovery and functional roles of GluRδ2 in the
cerebellum
The wealth of knowledge on the neural circuits inthe cerebellum makes the cerebellum an ideal system
to study the molecular mechanism of brain function.
By molecular cloning, we found a novel GluR subfamily named GluRδ. GluRδ2 was selectively localized in
cerebellar Purkinje cells. Furthermore, its intracellular
localization was restricted to the parallel fiberPurkinje cell synapses. The carboxyl terminus of
GluRδ2 interacted with delphilin containing a single
PDZ domain, formin homology (FH) domains and acoiled-coil structure. Analyses of GluRδ2 mutant mice
revealed that the GluRδ2 subunit was essential inmotor coordination and cerebellar LTD and in refinement and maintenance of Purkinje cell synapses.
We investigated eyeblink conditioning in GluRδ2mutant mice to elucidate its cerebellar cortical neuralmechanism, with reference to the temporal relationship of conditioned and unconditioned stimuli. In the
delay paradigm, in which a tone (CS) overlapped temporally with a periorbital shock (US), GluRδ2 mutant
mice exhibited a severe impairment in learning.
However, in the trace paradigm in which a stimulusfree trace interval up to 500 ms intervened between
the CS and US, GluRδ2 mutant mice learned as successfully as the wild-type mice even with 0 ms-traceinterval. We then examined the delay and trace eyeblink conditioning in NMDA receptor GluRε1 mutant
mice. In delay conditioning, GluRε1 mutant miceattained a normal level of the conditioned response(CR), although acquisition was a little slower than inwild-type mice. In contrast, GluRε1 mutant mice
exhibited severe impairment of the attained level of
the CR and disturbed temporal pattern of CR expression in trace conditioning with a longer trace intervalof 500 ms. These results suggest that neural substrates underlying eyeblink conditioning are different

depending on the temporal overlap of the conditioned
and unconditioned stimuli.

Current Research
These studies show that GluRs play key roles inmemory acquisition and neural pattern formation.
The memory signaling in the adult brain may sharethe common molecular mechanism with the activitydependent synapse refinement during neural development. To further investigate the molecular basis ofhigher brain function, we are developing the conditional gene targeting in the C57BL/6 mouse genetic
background and the molecular genetics of zebrafish.

Conditional gene targeting in C57BL/6 genetic
backgroundWe developed an efficient homologous recombination system using ES cells derived from C57BL/6
strain. For a cell type-specific and temporal regulationof gene targeting in the brain, we have generated
mouse lines that express Cre recombinase-progesterone receptor fusion (CrePR) gene specifically cerebellar granule cells, cerebellar Purkinje cells, hippocampal CA3 pyramidal neurons and striatal spiny
neurons. Crossing of target mice first with FLP mice
to remove the neo selection marker gene and thenwith Cre mice has yielded mutant mice lacking GluR
genes in specific neurons.
Molecular genetics of zebrafish
Elucidation of how the neural network is formed
and modulated is essential to understand how thebrain is functioning. The retinotectal projection andolfactory systems in transparent zebrafish are suitable
to analyze synapse formation in vivo. We are developing a novel strategy that visualizes and manipulates
developing neurons in vivo.

Future Prospects
Combination of molecular genetic approaches inmice and zebrafish will facilitate our understanding of
the mechanism of higher brain function at the molecular, cellular and neural network levels.

Research Grants
1. Grant-in-Aid for Scientific Research on Priority
Areas, The Ministry of Education, Culture, Sports,
Science and Technology (1995-1999)
2. Grant-in-Aid for Scientific Research (A), The
Ministry of Education, Culture, Sports, Science andTechnology (1996-1998)
3. CREST, Japan Science and Technology Corporation(1996-2001)

51

4. Grant-in-Aid for Scientific Research on Priority
Areas, The Ministry of Education, Culture, Sports,
Science and Technology (2000-)
5. SORST, Japan Science and Technology Corporation(2002-)

Select Publications
1. Mishina, M., Kurosaki, T., Tobimatsu, T., Morimoto,Y., Noda, M., Yamamoto, T., Terao, M., Lindstrom,J., Takahashi, T., Kuno, M. and Numa, S. Expressionof functional acetylcholine receptor from cloned
cDNAs. Nature 307, 604-608, 1984.
2. Mishina, M., Tobimatsu, T., Imoto, K., Tanaka, K.,
Fujita, Y., Fukuda, K., Kurasaki, M., Takahashi, H.,
Morimoto, Y., Hirose, T., Inayama, S., Takahashi, T.,
Kuno, M. and Numa, S. Location of functional
regions of acetylcholine receptor α-subunit by sitedirected mutagenesis. Nature 313, 364-369, 1985.
3. Takai, T., Noda, M., Mishina, M., Shimizu, S.,
Furutani, Y., Kayano, T., Ikeda, T., Kubo, T.,
Takahashi, H., Takahashi, T., Kuno, M. and Numa,
S. Cloning, sequencing and expression of cDNA for anovel subunit of acetylcholine receptor from calfmuscle. Nature 315, 761-764, 1985.
4. Sakmann, B., Methfessel, C., Mishina, M.,
Takahashi, T., Takai, T., Kurasaki, M., Fukuda, K.
and Numa, S. Role of acetylcholine receptor subunits
in gating of the channel. Nature 318, 538-543, 1985.
5. Mishina, M., Takai, T., Imoto, K., Noda, M.,
Takahashi, T., Numa, S., Methfessel, C. andSakmann, B. Molecular distinction between fetal andadult forms of muscle acetylcholine receptor. Nature
321, 406-411, 1986.
6. Kubo, T., Fukuda, K., Mikami, A., Maeda, A.,
Takahashi, H., Mishina, M., Haga, T., Haga, K.,
Ichiyama, A., Kangawa, K., Kojima, M., Matsuo, H.,
Hirose, T. and Numa, S. Cloning, sequencing andexpression of complementary DNA encoding themuscarinic acetylcholine receptor. Nature 323, 411416, 1986.
7. Imoto, K., Methfessel, C., Sakmann, B., Mishina, M.,
Mori, Y., Konno, T., Fukuda, K., Kurasaki, M., Bujo,
H., Fujita, Y. and Numa, S. Location of a δ-subunit
region determining ion transport through the acetylcholine receptor channel. Nature 324, 670-674, 1986.
8. Fukuda, K., Kubo, T., Akiba, I., Maeda, A., Mishina,
M. and Numa, S. Molecular distinction between
muscarinic acetylcholine receptor subtypes. Nature
327, 623-625, 1987.
9. Fukuda, K., Higashida, H., Kubo, T., Maeda, A.,
Akiba, I., Bujo, H., Mishina, M. and Numa, S.
Selective coupling with K+ currents of muscarinic
acetylcholine receptor subtypes in NG108-15 cells.
Nature 335, 355-358, 1988.
10. Imoto, K., Busch, C., Sakmann, B., Mishina, M.,
Konno, T., Nakai, J., Bujo, H., Mori, Y., Fukuda, K.
and Numa, S. Rings of negatively-charged aminoacids determine the acetylcholine receptor channel
conductance. Nature 335, 645-648, 1988.

52

11. Meguro, H., Mori, H., Araki, K., Kushiya, E.,
Kutsuwada, T., Yamazaki, M., Kumanishi, T.,
Arakawa, M., Sakimura, K. and Mishina, M.
Functional characterization of a heteromeric NMDA
receptor channel expressed from cloned cDNAs.
Nature 357, 70-74, 1992.
12. Kutsuwada, T., Kashiwabuchi, N., Mori, H.,
Sakimura, K., Kushiya, E., Araki, K., Meguro, H.,
Masaki, H., Kumanishi, T., Arakawa, M. andMishina, M. Molecular diversity of the NMDA receptor channel. Nature 358, 36-41, 1992.
13. Mori, H., Masaki, H., Yamakura, T. and Mishina, M.
Identification by mutagenesis of a Mg2+ block site of
the NMDA receptor channel. Nature 358, 673-675,
1992.
14. akimura, K., Kutsuwada, T., Ito, I., Manabe, T.,
Takayama, C., Kushiya, E., Yagi, T., Aizawa, S.,
Inoue, Y., Sugiyama, H. and Mishina, M. Reduced
hippocampal LTP and spatial learning in mice lacking NMDA receptor ε1 subunit. Nature 373, 151-155,
1995.
15. Kashiwabuchi, N., Ikeda, K., Araki, K., Hirano, T.,
Shibuki, K., Takayama, C., Inoue, Y., Kutsuwada, T.,
Yagi, T., Kang, Y., Aizawa, S. and Mishina, M.
Impairment of motor coordination, Purkinje cell
synapse formation and cerebellar long-term depression in GluRδ2 mutant mice. Cell 81, 245-252, 1995.
16. Kutsuwada, T., Sakimura, K., Manabe, T., Takayama,C., Katakura, N., Kushiya, E., Natsume, R.,
Watanabe, M., Inoue, Y., Yagi, T., Aizawa, S.,
Arakawa, M., Takahashi, T., Nakamura, Y., Mori, H.
and Mishina, M. Impairment of suckling response,
trigeminal neuronal pattern formation and hippocampal LTD in NMDA receptor ε2 subunit mutant
mice. Neuron 16, 333-344, 1996.
17. Takahashi, T., Feldmeyer, D., Suzuki, N., Onodera,
K., Cull-Candy, S. G., Sakimura, K. and Mishina, M.
Functional correlation of NMDA receptor ε subunits
expression with the properties of single-channel andsynaptic currents in the developing cerebellum. J.
Neurosci. 16, 4376-4382, 1996.
18. Ito, I., Futai, K., Katagiri, H., Watanabe, M.,
Sakimura, K., Mishina, M. and Sugiyama, H.
Synapse-selective impairment of NMDA receptor
functions in mice lacking NMDA receptor ε1 or ε2subunit. J. Physiol. 500, 401-408, 1997.
19. Taniguchi, M., Yuasa, S., Fujisawa, H., Naruse, I.,
Saga, S., Mishina, M. and Yagi, T. Disruption of semaphorin III/D gene causes severe abnormality inperipheral nerve projection. Neuron 19, 519-530,
1997.
20. Kurihara, H., Hashimoto, K., Kano, M., Takayama,C., Sakimura, K., Mishina, M., Inoue, Y. and
Watanabe, M. Impaired parallel fiber-Purkinje cell
synapse stabilization during cerebellar developmentof mutant mice lacking the glutamate receptor δ2
subunit. J. Neurosci. 17, 9613-9623, 1997.
21. Watanabe, M., Fukaya, M., Sakimura, K., Manabe,T., Mishina, M. and Inoue, Y. Selective scarcity of
NMDA receptor channel subunits in the stratum

lucidum (mossy fiber-recipient layer) of the hippocampal CA3 subfield. Eur. J. Neurosci. 10, 478-487,
1998.
22. Ando, H. and Mishina, M. Efficient mutagenesis ofzebrafish by a DNA cross-linking agent. Neurosci.
Lett. 244, 81-84, 1998.
23. Kohmura, N., Senzaki, K., Hamada, S., Kai, N.,
Yasuda, R., Watanabe, M., Ishii, H., Yasuda, M.,
Mishina, M. and Yagi, K. Diversity revealed by anovel family of cadherins expressed in neurons at
synaptic complex. Neuron 20, 1137-1151, 1998.
24. Kiyama, Y., Manabe, T., Sakimura, K., Kawakami, F.,
Mori, H. and Mishina, M. Increased thresholds forlong-term potentiation and contextual learning inmice lacking the NMDA-type glutamate receptor ε1
subunit. J. Neurosci. 18, 6704-6712, 1998.
25. Mori, H., Manabe, T., Watanabe, M., Satoh, Y.,
Suzuki, N., Toki, S., Nakamura, K., Yagi, T., Kushiya,E., Takahashi, T., Inoue, Y., Sakimura, K. andMishina, M. Role of the carboxyl-terminal region of
the GluRε2 subunit in synaptic localization of the
NMDA receptor channel. Neuron 21, 571-580, 1998.
26. Morikawa, E., Mori, H., Kiyama, Y., Mishina, M.,
Asano, T. and Kirino, T. Attenuation of focal
ischemic brain injury in mice deficient in the ε1(NR2A) subunit of NMDA receptor. J. Neurosci. 18,
9727-9732, 1998.
27. Hayashi, T., Umemori, H., Mishina, M. andYamamoto, T. The AMPA receptor interacts withand signals through the protein tyrosine kinase Lyn.
Nature 397, 72-76, 1999.
28. Furuyashiki, T., Fujisawa, K., Fujita, A., Madaule, P.,
Uchino, S., Mishina, M., Bito, H. and Narumiya, S.
Citron, a Rho-target, interacts with PSD-95/SAP-90 at
glutamatergic synapses in the thalamus. J. Neurosci.
19, 109-118, 1999.
29. Tsujita, M., Mori, H., Watanabe, M., Suzuki, M.,
Miyazaki, J. and Mishina, M. Cerebellar granule cellspecific and inducible expression of Cre recombinase
in the mouse. J. Neurosci. 19, 10318-10323, 1999.
30. Minami, T., Okuda-Ashitaka, E., Mori, H., Sakimura,K., Watanabe, M., Mishina, M. and Ito, S.
Characterization of nociceptin/orphanin FG-induced
pain responses in conscious mice: Neonatal capsaicin
treatment and N-methyl-D-aspartate receptor GluRεsubunit knockout mice. Neuroscience 97, 133-142,
2000.
31. Hironaka, K., Umemori, H., Tezuka, T., Mishina, M.
and Yamamoto, T. The protein-tyrosine phosphatase
PTPMEG interacts with glutamate receptor δ2 and εsubunits. J. Biol. Chem. 275, 16167-16173, 2000.
32. Matsuda, I. and Mishina, M. Identification of a juxtamembrane segment of the glutamate receptor δ2
subunit required for the plasma membrane localization. Biochem. Biophys. Res. Commun. 275, 565-571,
2000.
33. Nakamura, K., Manabe, T., Watanabe, M., Mamiya,T., Ichikawa, R., Kiyama, Y., Sanbo, M., Yagi, T.,
Inoue, Y., Nabeshima, T., Mori, H. and Mishina M.
Enhancement of hippocampal LTP, reference memo-

ry and sensorimotor gating in mutant mice lacking atelencephalon-specific cell adhesion molecule. Eur. J.
Neurosci. 13, 179-189, 2001.
34. Nakazawa, T., Komai, S., Tezuka, T., Hisatsune, C.,
Umemori, H., Semba, K., Mishina, M., Manabe, T.
and Yamamoto, T. Characterization of Fyn-mediatedtyrosine phosphorylation sites on GluRε2 (NR2B)
subunit of the N-methyl-D-aspartate receptor. J. Biol.
Chem. 276, 693-699, 2001.
35. Miyamoto, Y., Yamada, K., Noda, Y., Mori, H.,
Mishina, M. and Nabeshima, T. Hyperfunction of
dopaminergic and serotonergic neural systems inmice lacking NMDA receptor ε1 subunit. J. Neurosci.
21, 750-757, 2001.
36. Uchino, S., Nakamura, T., Nakamura, K., NakajimaIijima, S., Mishina, M., Kohsaka, S. and Kudo, Y.
Real-time two-dimensional visualization of ischemiainduced glutamate release from hippocampal slices.
Eur. J. Neurosci. 13, 670-678, 2001.
37. Kishimoto, Y., Kawahara, S., Mori, H., Mishina, M.
and Kirino, Y. Long-trace interval eyeblink conditioning is impaired in mutant mice lacking the NMDA
receptor subunit ε1. Eur. J. Neurosci. 13, 1221-1227,
2001.
38. Kishimoto, Y., Kawahara, S., Suzuki, M., Mori, H.,
Mishina, M. and Kirino, Y. Classical eyeblink conditioning in glutamate receptor subunit δ2 mutant
mice is impaired in the delay paradigm but not in the
trace paradigm. Eur. J. Neurosci. 13, 1249-1253,
2001.
39. Kitayama, K., Abe, M., Kakizaki, T., Honma, D.,
Natsume, R., Fukaya, M., Watanabe, M., Miyazaki,J., Mishina, M. and Sakimura, K. Purkinje cell-specific and inducible gene recombination system generated from C57BL/6 mouse ES cells. Biochem. Biophys.
Res. Commun. 281, 1134-1140, 2001.
40. Takeuchi, T., Kiyama, Y., Nakamura, K., Tsujita, M.,
Matsuda, I., Mori, H., Munemoto, Y., Kuriyama, H.,
Natsume, R., Sakimura, K. and Mishina, M. Roles of
the glutamate receptor ε2 and δ2 subunits in thepotentiation and prepulse inhibition of the acoustic
startle reflex. Eur. J. Neurosci. 14, 153-160, 2001.
41. Wainai, T., Takeuchi, T., Seo, N. and Mishina, M.
Regulation of acute nociceptive responses by the
NMDA receptor GluRε2 subunit. Neuroreport 12,
3165-3172, 2001.
42. Kishimoto, Y., Kawahara, S., Fujimichi, R., Mori, H.,
Mishina M., and Kirino Y. Impairment of eyeblinkconditioning in GluRδ2 mutant mice depends on thetemporal overlap between conditioned and unconditioned stimuli. Eur. J. Neurosci. 14, 1515-1521, 2001.
43. Hashimoto, K., Ichikawa, R., Takechi, H., Inoue, Y.,
Aiba, A., Sakimura, K., Mishina, M., Hashikawa, T.,
Konnerth, A., Watanabe, M., and Kano, M. Roles of
GluRδ2 and mGluR1 in climbing fiber synapse elimination during postnatal cerebellar development. J.
Neurosci. 21, 9701-9712, 2001.
44. Takatsuki, K., Kawahara, S., Mori, H., Mishina, M.
and Kirino, Y. Scopolamine impairs eyeblink condi-

53

tioning in the cerebellar LTD-deficient mice.
Neuroreport 13, 159-162, 2002.
45. Miyagi, Y., Yamashita, T., Fukaya, M., Sonoda, T.,Okuno, T., Yamada, K., Watanabe, M., Nagashima,Y., Aoki, I., Okuda, K., Mishina, M. and Kawamoto,S. Delphilin: a novel PDZ- and formin homologydomain containing protein, which synaptically colocalizes and interacts with glutamate receptor δ2 subunit. J. Neurosci. 22, 803-814, 2002.
46. Miyamoto, Y., Yamada, K., Noda, Y., Mori, H.,
Mishina, M. and Nabeshima, T. Lower sensitivity tostress and altered monoaminergic neuronal function
in mice lacking the NMDA receptor ε4 subunit. J.
Neurosci. 22, 2335-2342, 2002.
47. Takeuchi, T., Nomura, T., Tsujita, M., Suzuki, M.,
Fuse, T., Mori, H. and Mishina, M. Flp recombinasetransgenic mice of C57BL/6 strain for conditional
gene targeting. Biochem. Biophys. Res. Commun.
293, 953-957, 2002.
48. Yoshida, T., Ito, A., Matsuda, N. and Mishina, M.
Regulation by protein kinase A switching of axonal
pathfinding of zebrafish olfactory sensory neurons
through the olfactory placode-olfactory bulb boundary. J. Neurosci. 22, 4964-4972, 2002.
49. Uemura, T., Mori, H. and Mishina, M. Isolation andcharacterization of Golgi apparatus-specific GODZ
with the DHHC zinc finger domain. Biochem.
Biophys. Res. Commun. 296, 492-496, 2002.
50. Ichikawa, R., Miyazaki, T., Kano, M., Hashikawa, T.,
Tatsumi, H., Sakimura, K., Mishina, M., Inoue, Y.,
and Watanabe, M. Distal extension of climbing fiber
territory and multiple innervation caused by aberrant
wiring to adjacent spiny branchlets in cerebellar
Purkinje cells lacking glutamate receptor GluRδ2. J.
Neurosci. 22, 8487-8503, 2002.
51. Tokuoka, H., Yoshida, T., Matsuda, N. and Mishina,
M. (2002) Regulation by GSK-3β of the arborization
field and maturation of retinotectal projection inzebrafish. J. Neurosci., in press.

54